Method for the diagnosis, prognosis and treatment of lung cancer metastasis

ABSTRACT

The present invention relates to a method for the diagnosis or the prognosis of metastasis in lung cancer which comprises determining if the c-MAF gene is amplified in a primary tumor sample. Likewise, the invention also relates to a method for the diagnosis or the prognosis of metastasis in lung cancer, as well as to a method for determining the tendency to develop bone metastasis with respect to metastasis in other organs, which comprise determining the c-MAF gene expression level. Finally, the invention relates to the use of a c-MAF inhibitor as therapeutic target for treating the lung cancer.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 U.S.C. § 119(e) to U.S.Provisional Patent Application Ser. No. 61/656,372, filed on Jun. 6,2012, and incorporated herein by reference in its entirety.

REFERENCE TO SEQUENCE LISTING

The content of the electronically submitted sequence listing(“3190002PC01SEQListing.txt”, 48,392 bytes, created on Jun. 6, 2013)filed with the application is incorporated herein by reference in itsentirety.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates to the diagnosis or the prognosis ofmetastasis, in particular bone metastasis, in lung cancer based ondetermining if the c-MAF gene is amplified in a primary tumor sample.Likewise, the invention also relates to a method for the diagnosis orthe prognosis of metastasis in lung cancer, in particular/bonemetastasis, as well as to a method for designing a customized therapy ina subject with lung cancer, which comprises determining the c-MAF geneexpression level. Finally, the invention relates to the use of a c-MAFinhibitor as therapeutic target for the treatment of lung cancermetastasis, in particular bone metastasis.

Background Art

Metastasis, a complex process caused by elaborate interactions betweentumor cells and the surrounding normal tissues in different vitalorgans, accounts for 90 percent of all cancer deaths in patients withsolid tumors. The molecular and cellular mechanisms that lead primarytumors to form metastases must be understood in order to better addressthis major life-threatening problem. The identification of metastasisgenes and mechanisms is essential for understanding the basic biology ofthis lethal condition and its implications for clinical practice.Previous work provided a sense of the complexity of the metastasisprocess, but it failed to explain how and why metastasis occurs, whatmechanisms make metastasis a tissue-specific process, what events allowdormant metastases to become active and lethal many years after removalof a primary tumor, and what metastasis-mediating genes would eventuallyconstitute worthy diagnostic markers and therapeutic targets.

Lung Bone-Specific Metastasis

Lung cancer is a disease characterized by uncontrolled cell growth intissues of the lung. If left untreated, this growth can spread beyondthe lung in a process called metastasis into nearby tissue and,eventually, into other parts of the body. Most cancers that start inlung, known as primary lung cancers, are carcinomas that derive fromepithelial cells. Worldwide, lung cancer is the most common cause ofcancer-related death in men and women, and is responsible for 1.3million deaths annually, as of 2004. The most common symptoms areshortness of breath, coughing (including coughing up blood), and weightloss.

Lung cancers consist of four major types of lung cancer and multipleminor or rare forms. For clinico-pathological reasons they are oftendivided into the broad categories of small-cell lung cancer (SCLC), alsocalled oat cell cancer, and non-small-cell lung cancer (NSCLC). NSCLC isfurther divided into three major types, squamous, cell carcinoma (SCC),adenocarcinoma and large cell carcinomas. Because large cell carcinomasmight represent poorly or undifferentiated forms of the other types ofcancers, it is a vaguely defined entity, and criteria for diagnosis varywidely. The most common cause of lung cancer is long-term exposure totobacco smoke. Nonsmokers account for 15% of lung cancer cases, andthese cases are often attributed to a combination of genetic factors,radon gas, asbestos, and air pollution including secondhand smoke.

Similar to many other cancers, lung cancer is initiated by activation ofoncogenes or inactivation of tumor suppressor genes. Oncogenes are genesthat are believed to make people more susceptible to cancer.Proto-oncogenes are believed to turn into oncogenes when exposed toparticular carcinogens. Mutations in the K-ras proto-oncogene areresponsible for 10-30% of lung adenocarcinomas. The epidermal growthfactor receptor (EGFR) regulates cell proliferation, apoptosis,angiogenesis, and tumor invasion. Mutations and amplification of EGFRare common in non-small-cell lung cancer and provide the basis fortreatment with EGFR-inhibitors. Her2/neu is affected less frequently.Chromosomal damage can lead to loss of heterozygosity. This can causeinactivation of tumor suppressor genes. Damage to chromosomes 3p, 5q,13q, and 17p are particularly common in small-cell lung carcinoma. Thep53 tumor suppressor gene, located on chromosome 17p, is affected in60-75% of cases. Other genes that are often mutated or amplified arec-MET, NKX2-1, LKB1, PIK3CA, and BRAF.

Several genetic polymorphisms are associated with lung cancer. Theseinclude polymorphisms in genes coding for interleukin-1, cytochromeP450, apoptosis promoters such as caspase-8, and DNA repair moleculessuch as XRCC1. People with these polymorphisms are more likely todevelop lung cancer after exposure to carcinogens.

Classification

Lung cancers are classified according to histological type. Thisclassification has important implications for clinical management andprognosis of the disease. The vast majority of lung cancers arecarcinomas—malignancies that arise from epithelial cells. The two mostprevalent histological types of lung carcinoma, categorized by the sizeand appearance of the malignant cells seen by a histopathologist under amicroscope, are non-small-cell and small-cell lung carcinoma. Thenon-small-cell type is the most prevalent by far.

Cancer found outside of the lung may be determined to have arisen withinthe lung, as lung cancers that metastasize, i.e. spread, often retain acell marker profile that allow a pathologist to say, with a good deal ofcertainty, that the tumor arose from the lung, i.e. is a primary lungcancer. Primary lung cancers of adenocarcinoma histology typically havenuclear immunostaining with TTF-1.

Non-Small-Cell Lung Carcinoma

The non-small-cell lung carcinomas (NSCLC) are grouped together becausetheir prognosis and management are similar. There are three mainsub-types: squamous cell lung carcinoma; adenocarcinoma; and large-celllung carcinoma.

Accounting for 25% of lung cancers, squamous cell lung carcinoma usuallystarts near a central bronchus. A hollow cavity and associated necrosisare commonly found at the center of the tumor. Well-differentiatedsquamous cell lung cancers often grow more slowly than other cancertypes.

Adenocarcinoma accounts for 40% of non-small-cell lung cancers. Itusually originates in peripheral lung tissue. Most cases ofadenocarcinoma are associated with smoking; however, among people whohave never-smoked (“never-smokers”), adenocarcinoma is the most commonform of lung cancer. A subtype of adenocarcinoma, the bronchioloalveolarcarcinoma, is more common in female never-smokers, and may havedifferent responses to treatment.

Small-Cell Lung Carcinoma

Small-cell lung carcinoma (SCLC) is less common. It was formerlyreferred to as “oat-cell” carcinoma. Most cases arise in the largerairways (primary and secondary bronchi) and grow rapidly, becoming quitelarge. The small cells contain dense neurosecretory granules (vesiclescontaining neuroendocrinehormones), which give this tumor anendocrine/paraneoplastic syndrome association. While initially moresensitive to chemotherapy and radiation, it is often metastatic atpresentation, and ultimately carries a worse prognosis. Small-cell lungcancers have long been dichotomously staged into limited and extensivestage disease. This type of lung cancer is strongly associated withsmoking.

Prognosis

Prognostic factors in non-small-cell lung cancer include presence orabsence of pulmonary symptoms, tumor size, cell type (histology), degreeof spread (stage) and metastases to multiple lymph nodes, and vascularinvasion. For patients with inoperable disease, prognosis is adverselyaffected by poor performance status and weight loss of more than 10%.Prognostic factors in small-cell lung cancer include performance status,gender, stage of disease, and involvement of the central nervous systemor liver at the time of diagnosis.

For non-small-cell lung carcinoma (NSCLC), prognosis is generally poor.Following complete surgical resection of stage IA disease, five-yearsurvival is 61%. With stage IB disease, five-year survival is 57%. Thefive-year survival rate of patients with stage IV NSCLC is about 1%.

For small-cell lung carcinoma, prognosis is also generally poor. Theoverall five-year survival for patients with SCLC is about 5%. Patientswith extensive-stage SCLC have an average five-year survival rate ofless than 1%. The median survival time for limited-stage disease is 20months, with a five-year survival rate of 20%.

According to data provided by the National Cancer Institute, the medianage at diagnosis of lung cancer in the United States is 70 years, andthe median age at death is 72 years. In the US, people with medicalinsurance are more likely to have a better outcome.

Metastasis

Primary lung cancers themselves most commonly metastasize to the adrenalglands, liver, brain, and bone.

Skeletal metastasis occurs in advanced-stage lung cancer and they confera high level of morbidity. At first diagnosis of bone metastasis diseasetherapeutic intervention will usually involve systemic chemotherapy,radiotherapy and bisphophonates, which are mostly palliative optionswith the intention of reducing pain.

In healthy skeletal bone, an equal balance of new bone matrix formationand old bone matrix resorption is achieved via coordinated activity ofbone-degrading osteoclasts and bone-forming osteoblasts. Duringmetastasis bone disease, the normal balance of bone resorption andformation is disrupted by the homotypic and heterotypic cell-cellinteractions that occur between invading tumor cells, osteoblasts andostoclasts. Most patients with secondary bone tumors—including thoseassociated with lung cancer present with osteolytic lesions. Therefore,most treatment strategies in current use or under evaluation inmetastasis bone disease have been designed to protect the bone matrixfrom increased, bone degrading activity of osteoclasts. Bone metastasisosteolytic lesions of the lung are a common feature with those of thebreast, as opposed to prostate cancer. In the latter, an additionalcomplication present in castration-resistant prostate cancer andmetastasis bone disease are osteosclerotic lesions—also known asbone-forming or osteoblastic lesions—or a combination of both,osteolytic and osteosclerotic lesions—also referred to as mixed lesions.Osteosclerotic lesions are typified by bone deposits with multiplelayers of poorly organized type-I collagen fibrils that have a wovenappearance and reduced mechanical strength. Based on the commonphenotypic features of lung and breast cancer bone metastasis is likelythat the process underlines similar molecular mechanisms.

Lung cancer cells preserve, among each subtype,genome-aberration-induced transcriptional changes with high fidelity.The resulting dominant genes will reveal molecular events that predictthe metastatic outcome despite the existence of substantial genomic,transcriptional, translational, and biological heterogeneity in theoverall system. However, it is unknown whether the developmental historyof a cancer would result in different or common mediators ofsite-specific metastasis. Predisposing factors related to the cell oforigin may engender different rate-limiting barriers during metastasicprogression. The present patent aims to set the stage for a detailed newprognostic factor to predict metastasis to the bone and their potentialvalue as a therapeutic target.

The fact that most of the patients with solid tumor cancer die aftermetastasis means that it is crucial to understand the molecular andcellular mechanisms allowing a tumor to metastasize. Recent publicationshave demonstrated how the metastasis is caused by means of complex yetlittle known mechanisms and also how the different metastatic cell typeshave a tropism towards specific organs. These tissue specific metastaticcells have a series of acquired functions allowing them to colonizespecific organs.

However, there are no genetic markers, in the state of the art, whichallow the diagnosis and/or the prognosis of whether a patient whosuffers a specific lung cancer, such as NSCLC lung cancer, will or willnot suffer metastasis, thus a suitable therapy being able to be appliedto the subject suffering said cancer. Therefore, there is the need ofidentifying new markers which allow diagnosing the presence ofmetastasis in subjects suffering lung cancer and/or predicting theprobability of a subject suffering lung cancer to develop metastasis, inparticular bone metastasis. The identification of new prognosis factorswill serve as a guide in selecting the most suitable treatments.

Identification of markers that predict bone metastasis would provide apreventive therapeutic opportunity by imposing restrictions to thespreading and colonization of bone metastatic tissue by lung cells anddelay or transform a lethal condition. The inventors identified c-MAFand 16q22-24 genomic gain, as a bona fide associated lung cancer bonemetastasis gene and genomic alteration, in a preferred embodimentosteolytic prostate bone metastasis.

BRIEF SUMMARY OF THE INVENTION

The present inventors have determined that identifying the balance ofsignals that affect disseminated lung cancer cell metastasis, inparticular bone metastasis, would provide valuable information toestablish the prognosis of, and for preventive therapeutic interventionagainst, the disease. The inventors identified MAF and the 16q22-24genomic gain as playing a role in driving lung-to-bone metastasislesions, in particular osteolytic bone metastasis.

The present inventors have identified c-MAF as a marker associated witha greater tendency of lung cancer to cause metastasis and, particularly,bone metastasis. MAF over-expression appears to be due to anamplification of the locus 16q22-q24 in which the c-MAF gene is located.

c-MAF expression levels were studied in two patient cohorts. The firstcohort contained the expression profiles and the clinical notes ofprimary tumors from patients with lung cancer. The second patient cohortwas in the form of a tissue microarray composed of lung primary tumorbiopsies, including 9 tumors that developed metastasis to the bone, 16tumors that developed metastasis to other sites except bone and had aminimum clinical follow up of 5 years and 49 lung primary tumors thatnever developed metastasis with a minimum clinical follow up of 5 years,the c-MAF gene (mRNA) and protein expression in tumor cells and biopsycorrelates positively with different clinical parameters, included therecurrence, metastasis, bone metastasis and overall survival beingobserved. Furthermore, these studies showed the role of c-MAF as markerfor prognosis and as a target gene in lung cancer metastasis,particularly bone metastasis. Likewise, the inventors have associatedthe amplification of the locus 16q22-q24, including the c-MAF gene, withthe presence of metastasis, in particular bone metastasis, in subjectswith lung cancer.

Thus, in a first aspect, the invention relates to an vitro method forthe diagnosis of metastasis in a subject with lung cancer and/or theprognosis of the tendency to develop metastasis, in particular bonemetastasis, in a subject with lung cancer, which comprises:

-   -   (i) quantifying the c-MAF gene expression level or 16q22-24 copy        number gain in a tumor sample of said subject; and    -   (ii) comparing the expression level or copies previously        obtained with the expression level of said gene in a control        sample;        wherein if the expression level of said gene is increased or        genomic region amplified with respect to the expression level of        said gene in the control sample, then said subject has a        positive diagnosis for metastasis or a greater tendency to        develop metastasis, in particular bone metastasis.

In a second aspect, the invention relates to an in vitro method fordesigning a customized therapy for a subject with lung cancer, whichcomprises:

-   -   (i) quantifying the c-MAF gene expression level or 16q22-24 copy        number gain in a tumor sample of said subject; and    -   (ii) comparing the expression level previously obtained with the        expression level of said gene in a control sample;        wherein if the expression level is increased or 16q22-24 copy        number gained with respect to the expression level of said gene        in the control sample, then said subject is susceptible to        receive a therapy aiming to prevent an/or treat the metastasis.        In a particular aspect of this method, the subject is then        administered at least one therapy that prevents, inhibits and/or        treats the bone metastasis. If the expression level is not        increased or 16q22-24 copy number is not gained with respect to        said reference value, then said subject is not susceptible to        receive a therapy aiming to prevent, inhibit and/or treat the        bone metastasis. In a particular aspect of this method, the        subject is then not administered at least one therapy that        prevents, inhibits and/or treats the bone metastasis. In some        aspects, the reference value is the c-MAF gene expression level        in a sample of lung cancer from a subject who has not suffered        metastasis. In some aspects, the lung cancer, is SCL. In some        embodiments, the lung cancer is NSCLC. In some aspects, the lung        cancer is lung adenocarcinoma.

In a third aspect, the invention relates to an in vitro method fordesigning a customized therapy for a subject with lung cancer with bonemetastasis, which comprises:

-   -   (i) quantifying the c-MAF gene expression level or 16q22-24 copy        number gain in a bone metastatic tumor sample of said subject;        and    -   (ii) comparing the expression level obtained in step (i) with        the expression level of said gene in a control sample;        wherein if the c-MAF gene expression level or 16q22-24 copy        number gain is increased with respect to the expression level of        said gene in the control sample, then said subject is        susceptible to receive a therapy aiming to prevent the bone        degradation. In a particular aspect of this method, the subject        is then administered at least one therapy that prevents,        inhibits and/or treats the bone metastasis. If the c-MAF gene        expression level is not increased or 16q22-24 copy number is not        gained with respect to said reference value, then said subject        is not susceptible to receive a therapy for preventing the bone        degradation. In a particular aspect of this method, the subject        is then not administered at least one therapy that prevents,        inhibits and/or treats the bone metastasis.

In a fourth aspect, the invention relates to an in vitro method for thediagnosis of metastasis in a subject with lung cancer and/or for theprognosis of the tendency to develop metastasis in a subject with lungcancer which comprises determining if the c-MAF gene is amplified ortranslocated in a tumor tissue sample of said subject; wherein if saidgene is amplified with respect to a control sample, then said subjecthas a positive diagnosis for metastasis or a greater tendency to developmetastasis. In a particular aspect of this method, the subject is thenadministered at least one therapy that prevents or inhibits the bonemetastasis. In a particular aspect of this method, if the c-MAF gene isnot amplified or translocated in a tumor tissue sample of said subject,the subject is then not administered at least one therapy that prevents,inhibits and/or treats the bone metastasis. In a particular aspect, anabove average expression level of the c-MAF is indicative of anincreased risk of bone metastasis and this risk is proportional to thelevels of c-MAF expression. In a particular aspect, if the expressionlevel of the c-MAF is above the average plus one standard deviation withrespect to the average level, then it can be concluded that said subjecthas a greater tendency to develop early bone metastasis.

In another aspect, the invention relates to an in vitro method forpredicting the clinical outcome of a patient suffering lung cancer,which comprises determining if the c-MAF gene is amplified in a sampleof said subject relative to a reference gene copy number wherein anamplification of the c-MAF gene with respect to said reference gene copynumber is indicative of a poor clinical outcome. In a particular aspectof this method, the subject is then administered at least one therapythat prevents, inhibits and/or treats the bone metastasis. If suchamplification is not observed then the subject is then not administeredat least one therapy that prevents, inhibits and/or treats the bonemetastasis. In another embodiment of the seventh aspect, the inventionrelates to an in vitro method for predicting the clinical outcome of apatient suffering lung cancer which comprises determining if the c-MAFgene is translocated in a sample of said subject wherein a translocationof the c-MAF gene (e.g. t(14,16)) is indicative of a poor clinicaloutcome. In some embodiments, the invention relates to designing acustomized therapy for patients with the amplification or translocationof c-MAF. In some embodiments, the customized therapy is at least onetherapeutic drug that prevents, inhibits and/or treats the bonemetastasis.

In a fifth aspect, the invention relates to the use of a c-MAFinhibitory agent in the preparation of a medicinal product for treatingand/or preventing lung cancer metastasis, in particular bone metastasis.In another aspect, the invention relates to a c-MAF inhibitory agent foruse in the prevention of bone metastasis from lung cancer. In someaspects, the invention relates to a a c-MAF inhibitory agent for use inavoiding or preventing bone degradation.

In another aspect, the invention relates to the use of an agent capableof avoiding or preventing bone degradation in the preparation of amedicinal product for the treatment of bone metastasis in a subjectsuffering lung cancer and having elevated c-MAF levels or 16q22-24 copynumber gain in a metastatic tumor tissue sample with respect to acontrol sample.

In another aspect, the invention relates to a kit for predicting bonemetastasis of a lung cancer in a subject suffering from said cancer, thekit comprising: a) means for quantifying the expression level of c-MAFin a sample of said subject; and b) means for comparing the quantifiedlevel of expression of c-MAF in said sample to a reference c-MAFexpression level. The invention also relates to the use of such kit topredict bone metastasis of a lung cancer in a subject suffering fromsaid cancer. In one embodiment, the subject is then administered or notadministered at least one therapy that prevents, inhibits and/or treatsthe bone metastasis biased on the results of using the kit.

In another aspect, the invention relates to a kit for predicting bonemetastasis of a lung cancer in a subject suffering from said cancer, thekit comprising: a) means for quantifying the amplification of c-MAFgene, 16q23 or 16q22-24 locus amplification or translocation in a sampleof said subject; and b) means for comparing the amplified level of c-MAFgene, 16q23 or 16q22-24 locus amplification or translocation in saidsample to a reference. In another aspect, the invention relates to a kitfor predicting the clinical outcome of a subject suffering from bonemetastasis from a lung cancer, the kit comprising: a) means forquantifying the expression level of c-MAF or the 16q23 or 16q22-24 locusamplification or translocation in a sample of said subject; and b) meansfor comparing the quantified expression level of c-MAF or the 16q23 or16q22-24 locus amplification or translocation in said sample to areference c-MAF expression level or 16q23 or 16q22-24 locus. Theinvention also relates to the use of such kit to predict the clinicaloutcome of a subject suffering from bone metastasis from a lung cancer.In one embodiment, the subject is then administered or not administeredat least one therapy that prevents, inhibits and/or treats the bonemetastasis based on the results of using the kit.

In another aspect, the invention relates to a kit for determining atherapy for a subject suffering from lung cancer, the kit comprising: a)means for quantifying the expression level of c-MAF or the 16q23 or16q22-24 locus amplification or translocation in a sample of saidsubject; b) means for comparing, the quantified expression level ofc-MAF in said sample to a reference c-MAF expression level or 16q23 or16q22-24 locus; and c) means for determining a therapy for preventingand/or reducing bone metastasis in said subject based on the comparisonof the quantified expression level to the reference expression level.The invention also relates to the use of such kit to determine a therapyfor a subject suffering from lung cancer. In one embodiment, the subjectis then administered or not administered at least one therapy thatprevents, inhibits and/or treats the bone metastasis based on theresults of using the kit.

In another aspect, the invention relates to a kit comprising: i) areagent for quantifying the expression level of c-MAF or the 16q23 or16q22-24 locus amplification or translocation in a sample of a subjectsuffering from lung cancer, and ii) one or more c-MAF gene expressionlevel or the 16q23 or 16q22-24 locus amplification or translocationindices that have been predetermined to/correlate with the risk of bonemetastasis. The invention also relates to the use of such kit to predictbone metastasis of a lung cancer in a subject suffering from saidcancer. In one embodiment, the subject is then administered or notadministered at least one therapy that prevents, inhibits and/or treatsthe bone metastasis based on the results of using the kit.

In another aspect, the invention relates to an in vitro method fortyping a sample of a subject suffering from lung cancer, the methodcomprising:

-   -   a) providing a sample from said subject;    -   b) quantifying the expression level of c-MAF or the 16q23 or        16q22-24 locus amplification or translocation in said sample;    -   c) typing said sample by comparing the quantified expression        level of c-MAF or the 16q23 or 16q22-24 locus amplification or        translocation to a predetermined reference level of c-MAF        expression or 16q23 or 16q22-24 locus number of copies;        wherein said typing provides prognostic information related to        the risk of bone metastasis in said subject. In one embodiment,        the subject is administered or not administered at least one        therapeutic agent based on the prognostic information provided        by the typing.

In another aspect, the invention relates to a method for preventing orreducing the risk of bone metastasis in a subject suffering from lungcancer, said method comprising administering to said subject an agentthat prevents or reduces bone metastasis, wherein said agent isadministered in accordance with a treatment regimen determined fromquantifying the expression level of c-MAF or the 16q23 or 16q22-24 locusamplification or translocation in said subject.

In another aspect, the invention relates to a method of classifying asubject suffering from lung cancer into a cohort, the method comprising:a) determining the expression level of c-MAF or the 16q23 or 16q22-24locus amplification or translocation in a sample of said subject; b)comparing the expression level of c-MAF in said sample to apredetermined reference level of c-MAF expression or a normal genome;and c) classifying said subject into a cohort based on said expressionlevel of c-MAF or the 16q23 or 16q22-24 locus amplification ortranslocation in the sample. In one embodiment, the method is used toidentify patients predicted to have an earlier manifestation of symptomsof bone metastasis. In a particular aspect, the cohort is used forconducting a clinical trial.

BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES

FIG. 1: Gene expression of c-MAF in lung primary tumors correlatessignificantly with metastasis as its observed in kaplan meier plot andis highlighted by the HR and the p-value obtained.

FIG. 2: a) Kaplan-Meier plot depicting the probability of bonemetastasis free survival at any time in the overall population of lungcancer patients post primary tumor resection. b) Kaplan-Meier plotdepicting the probability of bone metastasis free survival at any timein lung cancer primary tumor biopsies according to c-MAF high or lowprotein expression categories measured by immunohistochemistry (IHC).The c-MAF expression cut of (20,000 Optical Density, O.D., Units) wasselected based on a receiving operating curve (ROC curve) as perstandard methods (Area under the curve, AUC, is 0.80).

FIG. 3: a) Kaplan-Meier plot depicting the probability of survival inthe overall population of lung cancer patients from the time of primarytumor resection. b) Kaplan-Meier plot depicting the probability ofsurvival in lung cancer primary according to c-MAF high or low proteinexpression categories. The c-MAF expression cut of (20,000 OpticalDensity, O.D., Units) was selected based on a receiving operating curve(ROC curve) based on bone metastasis at any time as per standardmethods.

DETAILED DESCRIPTION OF THE INVENTION

Methods for the Diagnosis and Prognosis of Lung Cancer Metastasis Basedon c-MAF Expression Levels

The inventors have identified that the c-MAF gene and protein isoverexpressed in lung cancer metastasis, and that the c-MAF expressionlevels in primary lung tumors are correlated to different clinicalparameters of lung cancer, particularly with recurrence and metastasisprobability. Thus, c-MAF overexpression is correlated with the onset oflung tumor metastasis in bone. Therefore, c-MAB can be used as a marker,for the diagnosis and/or prognosis of metastasis, in particular bonemetastasis, in a subject with lung cancer.

Thus in one aspect, the invention relates to an in vitro method for thediagnosis of metastasis in a subject with lung dancer and/or for theprognosis of the tendency to develop metastasis in a subject with lungcancer (hereinafter first method of the invention) which comprises

-   -   (i) quantifying the c-MAF gene expression level in a tumor        sample (e.g., lung tumor tissue, circulating lung tumor cell,        circulating lung tumor DNA) from said subject and    -   (ii) comparing the expression level previously obtained with the        expression level of said gene in a control sample,        wherein if the expression levels of said gene are increased with        respect to the expression levels of said gene in the control        sample, then said subject has a positive diagnosis for        metastasis or a greater tendency to develop metastasis, in a        preferred site bone metastasis.

The c-MAF gene (v-maf musculoaponeurotic fibrosarcoma oncogene homologue(avian) also known as MAF or MGC71685) is a transcription factorcontaining a leucine zipper which acts like a homodimer or aheterodimer. Depending on the DNA binding site, the encoded protein canbe a transcriptional activator or repressor. The DNA sequence encodingc-MAF is described in the NCBI database under accession number NG_016440(SEQ ID NO: 1). The coding sequence of c-MAF is set forth in SEQ ID NO:13. Two messenger RNA are transcribed from said DNA sequence, each ofthe which will give rise to one of the two c-MAF protein isoforms, the αisoform and the β isoform. The complementary DNA sequences for each ofsaid isoforms are described, respectively, in the NCBI database underaccession numbers NM_005360.4 (SEQ ID NO: 2) and NM_001031804.2 (SEQ IDNO: 3).

In the context of the present invention, “metastasis” is understood asthe propagation of a cancer from the organ where it started to adifferent organ. It generally occurs through the blood or lymphaticsystem. When the cancer cells spread and form a new tumor, the latter iscalled a secondary or metastatic tumor. The cancer cells forming thesecondary tumor are like those of the original tumor. If a lung, cancer,for example, spreads (metastasizes) to the bone, the secondary tumor isformed of malignant lung cancer cells. The disease in the bone ismetastatic lung cancer and riot bone cancer. In a particular embodimentof the method of the invention, the metastasis is lung cancer which hasspread (metastasized) to the bone.

In the present invention, “diagnosis of metastasis in a subject withlung cancer” is understood as identifying a disease (metastasis) bymeans of studying its signs, i.e., in the context of the presentinvention by means of increased c-MAF gene expression levels (i.e.,overexpression) in the lung cancer tumor-tissue with respect to acontrol sample.

In the present invention “prognosis of the tendency to developmetastasis in a subject with lung cancer” is understood as knowing basedon the signs if the lung cancer that said subject has will metastasizein the future. In the context of the present invention, the sign isc-MAF gene overexpression in tumor tissue.

The method of the invention comprises in a first step quantifying thec-MAF gene expression level in a tumor tissue sample from a subject.

In a preferred embodiment, the first method of the invention comprisesquantifying only the c-MAF gene expression level as a single marker,i.e., the method does not involve determining the expression level ofany additional marker.

As used herein, the term “subject” or “patient” refers to all animalsclassified as mammals and includes but is not limited to domestic andfarm animals, primates and humans, for example, human beings, non-humanprimates, cows, horses, pigs, sheep, goats, dogs, cats, or rodents.Preferably, the subject is a human man or woman of any age or race.

The terms “poor” or “good”, as used herein to refer to a clinicaloutcome, mean that the subject will show a favourable or unfavourableoutcome. As will be understood by those skilled in the art, such anassessment of the probability, although preferred to be, may not becorrect for 100% of the subjects to be diagnosed. The term, however,requires that a statistically significant portion of subjects can beidentified as having a predisposition for a given outcome. Whether aportion is statistically significant can be determined readily by theperson skilled in the art using various well known statistic evaluationtools, e.g., determination of confidence intervals, p-valuedetermination, Student's t-test, Mann-Whitney test, etc. Details arefound in Dowdy and Wearden, Statistics for Research, John Wiley & Sons,New York 1983. Preferred confidence intervals are at least about 50%, atleast about 60%, at least about 70%, at least about 80%, at least about90% at least about 95%. The p-values are, preferably, 0.05, 0.01, 0.005,or 0.0001 or less. More preferably, at least about 60 percent, at leastabout 70 percent, at least about 80 percent or at least about 90 percentof the subjects of a population can be properly identified by the methodof the present invention.

In the present invention “tumor sample” is understood as the sample(e.g., tumor tissue, circulating tumor cell, circulating tumor DNA)originating from the primary lung cancer tumor. Said sample can beobtained by conventional methods, for example biopsy, using methods wellknown by the persons skilled in related medical techniques. The methodsfor obtaining a biopsy sample include splitting a tumor into largepieces, or microdissection, or other cell separating methods known inthe art. The tumor cells can additionally be obtained by means ofcytology through aspiration with a small gauge needle. To simplifysample preservation and handling, samples can be fixed in formalin andsoaked in paraffin or first frozen and then soaked in a tissue freezingmedium such as OCT compound by means of immersion in a highly cryogenicmedium which allows rapid freezing.

As understood by the person skilled in the art, the gene expressionrevels can be quantified by measuring the messenger RNA levels of saidgene or of the protein encoded by said gene.

For this purpose, the biological sample can be treated to physically ormechanically break up the tissue or cell structure, releasing theintracellular, components into an aqueous or organic solution forpreparing nucleic acids. The nucleic acids are extracted by means ofcommercially available methods known by the person skilled in the art(Sambroock, J., et al., “Molecular cloning: a Laboratory Manual”, 3rded., Cold Spring Harbor Laboratory Press, N.Y., Vol. 1-3)

Thus, the c-MAF gene expression level can be quantified from the RNAresulting from the transcription of said gene (messenger RNA or mRNA)or, alternatively, from the complementary DNA (cDNA) of said gene.Therefore, in a particular embodiment of the invention, thequantification of the c-MAF gene expression levels comprises thequantification of the messenger RNA of the c-MAF gene or a fragment ofsaid mRNA, complementary DNA of the c-MAF gene or a fragment of saidcDNA or the mixtures thereof.

Virtually any conventional method can be used within the scope of theinvention for detecting and quantifying the mRNA levels encoded by thec-MAF gene or of the corresponding cDNA thereof. By way of non-limitingillustration, the mRNA levels encoded by said gene can be quantifiedusing conventional methods, for example, methods comprising mRNAamplification and the quantification of said mRNA amplification product,such as electrophoresis and staining, or alternatively, by Southern blotand using suitable probes, Northern blot and using specific probes ofthe mRNA of the gene of interest (c-MAF) or of the corresponding cDNAthereof, mapping with S1 nuclease, RT-PCR, hybridization, micro arrays,etc., preferably by means of real time quantitative PCR using a suitablemarker. Likewise, the cDNA levels corresponding to said mRNA encoded bythe c-MAF gene can also be quantified by means of using conventionaltechniques; in this case, the method of the invention includes a stepfor synthesizing the corresponding cDNA by means of reversetranscription (RT) of the corresponding mRNA followed by theamplification and quantification of said cDNA amplification product.Conventional methods for quantifying expression levels can be found, forexample, in Sambrook et al., 2001. (cited ad supra).

In a particular embodiment, the c-MAF gene expression levels arequantified by means of quantitative polymerase chain reaction (PCR) or aDNA or RNA array.

In addition, the c-MAF gene expression level can also be quantified bymeans of quantifying the expression levels of the protein encoded bysaid gene, i.e., the c-MAF protein (c-MAF) [NCBI, accession numberO75444], or any functionally equivalent variant of the c-MAF protein.There are two c-MAF protein isoforms, the α isoform (NCBI, NP_005351.2)made up of 403 amino acids (SEQ ID NO: 4) and the β isoform(NP_001026974.1) made up of 373 amino acids (SEQ ID NO: 5). The c-MAFgene expression level can be quantified by means of quantifying theexpression levels of any of the c-MAF protein isoforms. Thus, in aparticular embodiment, the quantification of the levels of the proteinencoded by the c-MAF gene comprises the quantification of the c-MAFprotein.

In the context of the present invention, “functionally equivalentvariant of the c-MAF protein” is understood as (i) variants of the c-MAFprotein (SEQ ID NO: 4 or SEQ ID NO: 5) in which one or more of the aminoacid residues are substituted by a conserved or non-conserved amino acidresidue (preferably a conserved amino acid residue), wherein suchsubstituted amino acid residue may or may not be one encoded by thegenetic code, or (ii) variants comprising an insertion or a deletion ofone or more amino acids and having the same function as the c-MAFprotein, i.e., to act as a DNA binding transcription factor. Variants ofthe c-MAF protein can be identified using methods based on the capacityof c-MAF for promoting in vitro cell proliferation as shown ininternational-patent application WO2605/046731 (hereby incorporated byreference in its entirety), based on the capacity of the so-calledinhibitor for blocking the transcription capacity of a reporter geneunder the control of cyclin D2 promoter or of a promoter containing thec-MAF responsive region (MARE or c-MAF responsive element) in cellsexpressing c-MAF as described in WO2008098351 (hereby incorporated byreference in its entirety), or based on the capacity of the so-calledinhibitor for blocking reporter gene expression under the control of theIL-4 promoter in response to the stimulation with PMA/ionomycin in cellsexpressing NFATc2 and c-MAF as described in US2009048117A (herebyincorporated by referenced its entirety).

The variants according to the invention preferably have sequencessimilarity with the amino acid sequence of any of the c-MAF proteinisoforms (SEQ ID NO: 4 or SEQ ID NO: 5) of at least 50%, at least 60%,at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98% or at least 99%. The degree of similarity between the variantsand the specific c-MAF protein sequences defined previously isdetermined using algorithms and computer processes which are widelyknown by the persons skilled in the art. The similarity between twoamino acid sequences is preferably determined using the BLASTP algorithm[BLAST Manual, Altschul, S., et al., NCBI NLM NIH Bethesda, Md. 20894,Altschul, S., et al., J. Mol. Biol. 215: 403-410 (1990)].

The c-MAF protein expression level can be quantified by any conventionalmethod which allows detecting and quantifying said protein in a samplefrom a subject. By way of non-limiting illustration, said protein levelscan be quantified, for example, by using antibodies with c-MAF bindingcapacity (or a fragment thereof containing an antigenic determinant) andthe subsequent quantification of the complexes formed. The antibodiesused in these assays may or may not be labeled. Illustrative examples ofmarkers that can be used include radioactive isotopes, enzymes,fluorophores, chemiluminescence reagents, enzyme substrates orcofactors, enzyme inhibitors; particles, dyes, etc. There is a widerange of known assays that can be used in the present invention whichuse unlabeled antibodies (primary antibody) and labeled antibodies(secondary antibody); these techniques include Western-blot or Westerntransfer, ELISA (enzyme-linked immunosorbent assay), RIA(radioimmunoassay), competitive EIA (competitive enzyme immunoassay),DAS-ELISA (double antibody sandwich ELISA), immunocytochemical andimmunohistochemical techniques, techniques based on the use of proteinmicroarrays or biochips including specific antibodies, or assays basedon colloidal precipitation in formats such as dipsticks. Other ways fordetecting and quantifying said c-MAF protein include affinitychromatography techniques, ligand binding assays, etc. When animmunological method is used, any antibody or reagent that is known tobind to the c-MAF protein with a high affinity can be used for detectingthe amount thereof. Nevertheless, the use of an antibody, for example,polyclonal sera, supernatants of hybridomas or monoclonal antibodies,antibody fragments, Fv, Fab, Fab′ and F(ab′)2, scFv, humanizeddiabodies, triabodies, tetrabodies, nanobodies, alphabodies, stapledpeptides, cyclopeptides and antibodies. There are commercial anti-c-MAFprotein antibodies on the market which can be used in the context of thepresent invention, such as for example antibodies ab427, ab55502,ab55502, ab72584, ab76817, ab77071 (Abcam plc, 330 Science Park,Cambridge CB4 0FL, United Kingdom), the O75444 monoclonal antibody(Mouse Anti-Human MAF Azide free Monoclonal antibody, Unconjugated,Clone 6b8) of AbD Serotec, etc. There are many commercial companiesoffering anti-c-MAF antibodies, such as Abnoya Corporation, BethylLaboratories, Santa Cruz Biotechnology, Bioworld Technology, GeneTex,etc.

In a particular embodiment, the c-MAF protein levels are quantifiedmeans of western blot, immunohistochemistry, ELISA of a protein array.

In another particular, embodiment, the c-MAF protein levels arequantified from exosomes or circulating DNA. Exosomes are 40-100 nmmembrane vesicles secreted by most cell types in vivo and in vitro.Exosomes form in a particular population of endosomes, calledmultivesicular bodies (MVBs) by inward budding into the lumen of thecompartment. Upon fusion of MVBs with the plasma membrane, theseinternal vesicles are secreted. Exosomes can be isolated from diversecell lines or body fluids by several methods well known in the art(Théry C. et al., Curr Protoc Cell Biol. 2006 April; Chapter 3 Unit3.22) (the entire contents of which are incorporated by referenceherein). Several commercial kits are available for the isolation ofexosomes such as ExoQuick™ or ExoTest™.

The first method of the invention comprises in a second step comparingthe c-MAF gene expression level obtained in the tumor sample (includingbut not limited to a primary tumor biopsy, circulating tumor cells andcirculating tumor DNA) from the subject with the expression level ofsaid gene in a control sample.

Once the c-MAF gene expression level in a tumor tissue sample, acirculating tumor cell or circulating tumor DNA from a subject with lungcancer has been measured and compared with the control sample, if theexpression level of said gene are increased with respect to itsexpression level in the control sample, then it can be concluded thatsaid subject has a positive diagnosis for metastasis or a greatertendency to develop metastasis.

The determination of the c-MAF gene expression level must be correlatedwith values of a control sample or reference sample. Depending on thetype of tumor to be analyzed, the exact nature of the control sample mayvary. Thus, in the event that a diagnosis is to be evaluated, then thereference sample is a tumor tissue sample from a subject with lungcancer that has not metastasized or that corresponds to the median valueof the c-MAF gene expression levels measured in a tumor tissuecollection in biopsy samples from subjects with lung cancer which havenot metastasized.

Said reference sample is typically obtained by combining equal amountsof samples from a subject population. Generally, the typical referencesamples will be obtained from subjects who are clinically welldocumented and in whom the absence of metastasis is well characterized.In such samples, the normal concentrations (reference concentration) ofthe biomarker (c-MAF gene) can be determined, for example by providingthe mean concentration over the reference population. Variousconsiderations are taken into account when determining the referenceconcentration of the marker. Among such considerations are the age,weight, sex, general physical condition of the patient and the like. Forexample, equal amounts of a group of at least 2, at least 10, at least100 to preferably more than 1000 subjects, preferably classifiedaccording to the foregoing considerations, for example according tovarious age categories, are taken as the reference group. The samplecollection from which the reference level is derived will preferably beformed by subjects suffering from the same type of cancer as the patientobject of the study (e.g., lung cancer). Similarly, the reference valuewithin a cohort of patients can be established using a receivingoperating curve (ROC) and measuring the area under the curve for allsensitivity and specificity pairs to determine which pair provides thebest values and to which reference value corresponds.

Once this median or reference value has been established, the level ofthis marker expressed in tumor tissues from patients with this medianvalue can be compared and thus be assigned to the “increased” expressionlevel. Due to the variability among subjects (for example, aspectsreferring to age, race, etc.) it is very difficult (if not virtuallyimpossible) to established absolute reference, values of c-MAFexpression. Thus, in particular embodiments the reference values for“increased” or “reduced” expression of the c-MAF expression aredetermined by calculating the percentiles by conventional means whichinvolves performing assays in one or several samples isolated fromsubjects whose disease is well documented by any of the methodsmentioned above the c-MAF expression levels. The “reduced” levels ofc-MAF can then preferably be assigned to samples wherein the c-MAFexpression levels are equal to or lower than 50^(th) percentile in thenormal population including, for example, expression levels equal to orlower than the 60^(th) percentile in the normal population, equal to orlower than the 70^(th) percentile in the normal population, equal to orlower than the 80^(th) percentile in the normal population, equal to orlower than the 90^(th) percentile in the normal population, and equal toor lower than the 95^(th) percentile in the normal population. The“increased” c-MAF gene expression levels can then preferably be assignedto samples wherein the c-MAF gene expression levels are equal to orgreater than the 50^(th) percentile in the normal population including,for example, expression levels equal to or greater than the 60^(th)percentile in the normal population, equal to or greater than the70^(th) percentile in the normal population, equal to or greater thanthe 80^(th) percentile in the normal population, equal to or greaterthan the 90^(th) percentile in the normal population, and equal to orgreater than the 95^(th) percentile in the normal population.

In the present invention “increased expression levels” is understood asthe expression level when it refers to the levels of the c-MAF genegreater than those in a reference sample or control sample.Particularly, a sample can be considered to have high c-MAF expressionlevels when the expression levels in the reference sample are at least1.1 times, 1.5 times, 5 times, 10 times, 20 times, 30 times, 40 times,50 times, 60 times, 70 times, 80 times, 90 times, 100 times or even morewith respect to the sample isolated from the patient.

In the context of the present invention, it is understood that “asubject has a positive diagnosis for metastasis” when the lung cancersuffered by said subject has metastasized to other organs of the body,in a particular embodiment, to the bone.

In a yet more preferred embodiment, the metastasis to bone is anosteolytic bone metastasis. As used herein, the expression “osteolyticbone metastasis” refers to a type of metastasis in which bone resorption(progressive loss of the bone density) is produced in the proximity ofthe metastasis resulting from the stimulation of the osteoclast activityby the tumor cells and is characterized by severe pain, pathologicalfractures, hypercalcaemia, spinal cord compression and other syndromesresulting from nerve compression.

On the other hand, it is understood in the present invention that “asubject has a greater tendency to develop metastasis” when theprobabilities that the lung cancer suffered by the subject willmetastasize in the future are high.

The person skilled in the art will understand that the prediction of thetendency for a primary lung tumor to metastasize is not intended to becorrect for all the subjects to be identified (i.e., for 100% of thesubjects). Nevertheless, the term requires enabling the identificationof a statistically significant part of the subjects (for example, acohort in a cohort study). Whether a part is statistically significantcan be determined in a simple manner by the person skilled in the artusing various well known statistical evaluation tools, for example, thedetermination of confidence intervals, determination of p values,Student's T test Mann-Whitney test, etc. Details are provided in Dowdyand Wearden, Statistics for Research, John Wiley and Sons, New York1983. The preferred confidence intervals are at least 90%, at least 95%,at least 97%, at least 98% or at least 99%. The p values are preferably0.1, 0.05, 0.01, 0.005 or 0.0001. More preferably, at least 60%, atleast 70%, at least 80% or at least 90% of the subjects of a populationcan be suitably identified by the method of the present invention.

As used herein, “agent for avoiding or preventing bone degradation”refers to any molecule capable of preventing, inhibiting, treating,reducing, or stopping bone degradation either by stimulating theosteoblast proliferation or inhibiting the osteoclast proliferation orfixing the bone structure.

As used herein, a “c-MAF inhibitory agent” refers to any moleculecapable of completely or partially inhibiting the c-MAF gene expression,both by preventing the expression product of said gene from beingproduced (interrupting the c-MAF gene transcription and/or blocking thetranslation of the mRNA coming from the c-MAF gene expression) and bydirectly inhibiting the c-MAF protein activity, c-MAF gene expressioninhibitors can be identified using methods based on the capacity, of theso-called inhibitor to block the capacity of c-MAF to promote the invitro cell proliferation, such as shown in the international patentapplication WO2005/046731 (the entire contents of which are herebyincorporated by reference), based on the capacity of the so-calledinhibitor to block the transcription capacity of a reporter gene underthe control of the-cyclin D2 promoter or of a prompter containing thec-MAF response region (MARE or c-MAF responsive element) in cells whichexpress c-MAF such as described in WO2008098351 (the entire contents ofwhich are hereby incorporated by reference) or based on the capacity ofthe so-called inhibitor to block the expression of a reporter gene underthe control of the IL-4 promoter in response to the stimulation withPMA/ionomycin in cells which express NFATc2 and c-MAF such as describedin US2009048117A (the entire contents of which is hereby incorporated byreference).

As used herein, Mammalian target of rapamycin (mTOR) or “mTor” refers tothose proteins that correspond to EC 2.7.11.1 mTor enzymes areserine/threonine protein kinases and regulate cell proliferation, cellmotility, cell growth, cell survival, and transcription.

As used herein, an “mTor inhibitor” refers to any molecule capable ofcompletely or partially inhibiting the mTor gene expression, both bypreventing the expression product of said gene from being produced(interrupting the mTor gene transcription and/or blocking thetranslation of the mRNA coming from the mTor gene expression) and bydirectly inhibiting the mTor protein activity. mTor inhibitors, includeinhibitors that have one or more targets in addition to inhibition ofmTor activity.

As used herein, “Src” refers to those proteins that correspond to EC2.7.10.2. Src is a non-receptor tyrosine kinase and a proto-oncogene.Src may play a role in cell growth and embryonic development.

As used herein, a “Src inhibitor” refers to any molecule capable ofcompletely or partially inhibiting the Src gene expression, both bypreventing the expression product of said gene from being produced(interrupting the Src gene transcription and/or blocking the translationthe mRNA coming from the Src gene expression) and by directly inhibitingthe Src protein activity.

As used herein, “Prostaglandin-endoperoxide synthase 2”;“cyclooxygenase-2” or “COX-2” refers to those proteins that correspondto EC 1.14.99.1. COX-2 is responsible for converting arachidonic acid toprostaglandin endoperoxide H2.

As used herein, a “COX-2 inhibitor” refers to any molecule capable ofcompletely or partially inhibiting the COX-2 gene expression, both bypreventing the expression product of said gene from being produced(interrupting the COX-2 gene transcription and/or blocking thetranslation of the mRNA coming from the COX-2 gene expression) and bydirectly inhibiting the COX-2 protein activity.

As used herein “outcome” or “clinical outcome” refers to the resultingcourse of disease and/or disease progression and can be characterizedfor example by recurrence, period of time until recurrence, metastasis,period of time until metastasis, number of metastases, number of sitesof metastasis and/or death due to disease. For example a good clinicaloutcome includes cure, prevention of recurrence, prevention ofmetastasis and/or survival within a fixed period of time (withoutrecurrence), and a poor clinical outcome includes disease progression,metastasis and/or death within a fixed period of time.

“Predicting”, as used herein, refers to the determination of thelikelihood that the subject suffering lung cancer will developmetastasis to a distant organ. As used herein, “good prognosis”indicates that the subject is expected (e.g. predicted) to surviveand/or have no, or is at low risk of having, recurrence or distantmetastases within a set time period. The term “low” is a relative termand, in the context of this application, refers to the risk of the “low”expression group with respect to a clinical outcome (recurrence, distantmetastases, etc.). A “low” risk can be considered as a risk lower thanthe average risk for an heterogeneous cancer patient population. In thestudy of Paik et al. (2004), an overall “low” risk of recurrence wasconsidered to be lower than 15 percent. The risk will also vary infunction of the time period. The time period can be, for example, fiveyears, ten years, fifteen years or even twenty years after initialdiagnosis of cancer or after the prognosis was made.

As used herein, “poor prognosis” indicates that the subject is expectede.g. predicted to not survive and/or to have, or is at high risk ofhaving, recurrence or distant metastases within a set time period. Theterm “high” is a relative term and, in the context of this application,refers to the risk of the “high” expression group With respect to aclinical outcome (recurrence, distant metastases, etc.). A “high” riskcan be considered as a risk higher than the average risk for aheterogeneous cancer patient population. In the study of Paik et al.(2004), an overall “high” risk of recurrence was considered to be higherthan 15 percent. The risk will also vary in function of the time period.The time period can be, for example, five years, ten years, fifteenyears or even twenty years of initial diagnosis of cancer or after theprognosis was made.

“Reference value”, as used herein, refers to a laboratory value used asa reference for values/data obtained by laboratory examinations ofpatients or samples collected from patients. The reference value orreference level can be an absolute value; a relative value; a value thathas an upper and/or lower limit; a range of values; an average value; amedian value, a mean value, or a value as compared to a particularcontrol or baseline value. A reference value can be based on anindividual sample value, such as for example, a value obtained from asample from the subject being tested, but at an earlier point in time.The reference value can be based on a large number of samples, such asfrom a population of subjects of the chronological age matched group, orbased on a pool of samples including or excluding the sample to betested.

The term “treatment”, as used herein, refers to any type of therapy,which aims at terminating, preventing, ameliorating or reducing thesusceptibility to a clinical condition as described herein. In apreferred embodiment, the term treatment relates to prophylactictreatment (i.e. a therapy to reduce the susceptibility to a clinicalcondition), of a disorder or a condition as defined herein. Thus,“treatment,” “treating,” and their equivalent terms refer to obtaining adesired pharmacologic or physiologic effect, covering any treatment of apathological condition or disorder in a mammal, including a human. Theeffect may be prophylactic in terms of completely or partiallypreventing a disorder, or symptom thereof and/or may be therapeutic interms of a partial or complete cure for a disorder and/or adverse effectattributable to the disorder. That is, “treatment” includes (1)preventing the disorder from occurring or recurring in a subject, (2)inhibiting the disorder, such as arresting its development, (3) stoppingor terminating the disorder or at least symptoms associated therewith,so that the host no longer suffers from the disorder or its symptoms,such as causing regression of the disorder or its symptoms, for example,by restoring or repairing a lost, missing or defective function, orstimulating an inefficient process, or (4) relieving, alleviating, orameliorating the disorder, or symptoms associated therewith, whereameliorating is used in a broad senseto refer to at least a reduction inthe magnitude of a parameter, such as inflammation, pain, or immunedeficiency.

As used herein, “sample” or “biological sample” means biologicalmaterial isolated from a subject. The biological sample may contain anybiological material suitable for determining the expression level of thec-MAF gene. The sample can be isolated from any suitable biologicaltissue or fluid such as, for example, tumor tissue, blood, blood plasma,serum, urine or cerebral spinal fluid (CSF).

As used herein, the term “expression level” of a gene as used hereinrefers to the measurable quantity of gene product produced by the genein a sample of the subject, wherein the gene product can be atranscriptional product or a translational product. Accordingly, theexpression level can pertain to a nucleic acid gene product such as mRNAor cDNA or a polypeptide gene product. The expression level is derivedfrom a subject's sample and/or a reference sample or samples, and canfor example be detected de novo or correspond to a previousdetermination. The expression level can be determined or measured, forexample, using microarray methods, PCR methods (such as qPCR), and/orantibody based methods, as is known to a person of skill in the art.

As used herein, the term “gene copy number” refers to the copy number ofa nucleic acid molecule in a cell. The gene copy number includes thegene copy number in the genomic (chromosomal) DNA of a cell. In a normalcell (non-tumoral cell), the gene copy number is normally two copies(one copy in each member of the chromosome pair). The gene copy numbersometimes includes half of the gene copy number taken from samples of acell population.

“Increased expression level” is understood as the expression level whenit refers to the levels of the c-MAF gene greater than those in areference sample or control sample. This increased levels can be causedwithout excluding other mechanisms by a gene or 16q23 or 16q22-24chromosomal locus amplification or translocation. Particularly, a samplecan be considered to have high c-MAF expression level when theexpression level in the sample isolated from the patient is at leastabout 1.1 times, 1.2 times, 1.3 times, 1.4 times, 1.5 times, 2 times, 3times, 4 times, 5 times, 10 times, 20 times, 30 times, 40 times, 50times, 60 times, 70 times, 80 times, 90 times, 100 times or even morewith respect to the reference or control.

“Probe”, as used herein, refers to an oligonucleotide sequence that iscomplementary to a specific nucleic acid sequence of interest. In someembodiments, the probes may be specific to regions of chromosomes whichare known to undergo translocations. In some embodiments, the probeshave a specific label or tag. In some embodiments, the tag is afluorophore. In some embodiments, the probe is a DNA in situhybridization probe whose labeling is based on the stable coordinativebinding of platinum to nucleic acids and proteins. In some embodiments,the probe is described in U.S. patent application Ser. No. 12/067,532and U.S. patent application Ser. No. 12/181,399, which are incorporatedherein by reference in their entirety, or as described in Swennenhuis etal. “Construction of repeat-free fluorescence in situ hybridizationprobes” Nucleic Acids Research 40(3): e20 (2012).

“Tag” or “label”, as used herein, refers to any physical molecule whichis directly or indirectly associated with a probe, allowing the probe orthe location of the probed to be visualized, marked, or otherwisecaptured.

“Translocation”, as used herein, refers to the exchange of chromosomalmaterial in unequal or equal amounts between chromosomes. In some cases,the translocation is on the same chromosome. In some cases, thetranslocation is between different chromosomes. Translocations occur ata high frequency in many types, of cancer, including breast cancer andleukemia. Translocations can be either primary reciprocal translocationsor the more complex secondary translocations. There are several primarytranslocations that involve the immunoglobin heavy chain (IgH) locusthat are believed to constitute the initiating event in many cancers.(Eychène, A., Rocques, N., and Puoponnot, C., A new MAFia in cancer.2008. Nature Reviews: Cancer. 8:683-693.)

“Polyploid” or “polyploidy”, as used herein, indicates that the cellcontains more than two copies of a gene, of interest. In some instances,the gene of interest is MAF. In some embodiments, polyploidy isassociated with an accumulation of expression of the gene of interest.In some embodiments, polyploidy is 32 associated with genomicinstability. In some embodiments, the genomic instability may lead tochromosome translocations.

“Whole genome sequencing”, as used herein, is a process by which theentire genome of an organism is sequenced at a single time. See, e.g.,Ng., P. C. and Kirkness, E. F., Whole Genome Sequencing. 2010. Methodsin Molecular Biology. 628:215-226.

“Exome sequencing”, as used herein, is a process by which the entirecoding region of the DNA of an organism is sequenced. In exomesequencing, the mRNA is sequenced. The untranslated regions of thegenome are not included in exome sequencing. See, e.g., Choi, M. et al.,Genetic diagnosis by whole exome capture and massively parallel DNAsequencing. 2009. PNAS. 106(45): 19096-19101.

“Tumor tissue sample” is understood as the tissue sample originatingfrom the lung cancer tumor, including but hot limited to circulatingtumor cells and circulating tumor DNA. Said sample can be obtained byconventional methods, for example biopsy, using methods well known bythe persons skilled in related medical techniques.

“Osteolytic bone, metastasis” refers to a type of metastasis in whichbone resorption (progressive loss of the bone density) is produced inthe proximity of the metastasis resulting from the stimulation of theosteoclast activity by the tumor cells and is characterized by severepain, pathological fractures, hypercalcemia, spinal cord compression andother syndromes resulting from nerve compression.

Method for Designing Customized Therapy of the Invention in Patientswith Lung Tumors

As is known in the state of the art, the treatment to be administered toa subject suffering from cancer depends on whether the latter is amalignant tumor, i.e., whether it has high probabilities of undergoingmetastasis, or whether the latter is a benign tumor. In the firstassumption, the treatment of choice is a systemic treatment such aschemotherapy and in the second assumption, the treatment of choice is alocalized treatment such as radiotherapy.

Therefore, as described in the present invention, given that the c-MAFgene overexpression in lung cancer cells is related to the presence ofmetastasis, the c-MAF gene expression levels allow making decisions interms of the most suitable therapy for the subject suffering saidcancer.

Thus, in another aspect the invention relates to an in vitro method fordesigning a customized therapy for a subject with lung cancer, whichcomprises

-   -   (i) quantifying the c-MAF gene expression level in a tumor        sample of said subject and    -   (ii) comparing the expression level previously obtained with the        expression level of said gene in a control sample,        wherein if the expression levels are increased with respect to        the expression levels of said gene in the control sample, then        said subject is susceptible to receive a therapy aiming to        prevent and/or treat the metastasis. In a particular aspect of        the invention, the subject is then administered at least one        therapy that prevents, inhibits and/or treats the bone        metastasis.        wherein if the c-MAF gene expression level is not increased with        respect to said reference value, then said subject is not        susceptible to receive a therapy for preventing the bone        degradation. In a particular aspect of this method, the subject        is then not administered at least one therapy that prevents,        inhibits and/or treats the bone metastasis.

In a particular embodiment, the metastasis is a bone metastasis. In amore preferred embodiment, the bone metastasis is osteolytic metastasis.

The terms and expressions “subject”, “lung cancer”, “tumor sample”,“metastasis”, “determination of expression levels”, “c-MAF gene”,“increased expression levels” and “control sample” have been describedin detail in relation lo the first method of the invention and areequally applicable to the second and third method of the invention.

The second method of the invention comprises in a first step quantifyingthe c-MAF gene expression level in a tumor sample in a subject sufferingfrom lung cancer.

In a preferred embodiment, the second method of the invention comprisesquantifying only the c-MAF gene expression level as a single marker,i.e., the method does not involve determining the expression level ofany additional marker.

In the case of the second method of the invention the sample is aprimary tumor tissue sample of the subject. In a second step, the c-MAFgene expression level obtained in the tumor sample of the subject iscompared with the expression level of said gene in a control sample. Thedetermination of the c-MAF gene expression levels must be related tovalues of a control sample or reference sample. Depending on the type oftumor to be analyzed, the exact nature of the control sample may vary.Thus preferably the reference sample is a tumor tissue sample of subjectwith lung cancer that has not metastasized or that correspond to themedian value of the c-MAF gene expression levels measured in a tumortissue collection in biopsy samples of subjects with lung cancer whichhas not metastasized.

In yet another embodiment, an expression level of c-MAF which is abovethe average indicates increased risk of bone metastasis, the risk beingproportional to the levels of c-MAF expression. Thus, the risk of bonemetastasis in a subject suffering lung cancer is dose-dependent.

Once the c-MAF gene expression levels in the sample have been measuredand compared with the control sample, levels of said gene are increasedwith respect to their expression levels in the control sample, then itcan be concluded that said subject is susceptible to receiving therapyaiming to prevent (if the subject has yet to undergo metastasis) and/ortreat metastasis (if the subject has already experienced metastasis). Ifsuch increased expression is not observed then the subject is notadministered at least one therapy that prevents, inhibits and/or treatsthe bone metastasis.

When the cancer has metastasized, systemic treatments including but notlimited to chemotherapy, hormone treatment, immunotherapy, or acombination thereof are used. Additionally, radiotherapy and/or surgerycan be used. The choice of treatment generally depends on the type ofprimary cancer, the size, the location of the metastasis, the age, thegeneral health of the patient and the types of treatments usedpreviously.

The systemic treatments are those that reach the entire body:

-   -   Chemotherapy is the use of medicaments to destroy cancer cells.        The medicaments are generally administered through oral or        intravenous route. Sometimes, chemotherapy is used together with        radiation treatment. Suitable chemotherapeutic treatments for        lung cancer include, without limitation, anthracyclines        (doxorubicin, epirubicin, pegylated liposomal doxorubicin),        Taxanes (paclitaxel, docetaxel, albumin nano-particle bound        paclitaxel), 5-fluorpuracil (continuous infusion 5-FU,        capecitabine), Vinca alkaloids (vinorelbine, vinblastine),        Gemcitabine, Platinum salts (cisplatin, carboplatin),        cyclophosphamide, Etoposide and combinations of one or more of        the above such as Cyclophosphamide/anthracycline        +/−5-fluorouracil regimens (such as doxorubicin/cyclophosphamide        (AC), epirubicin/cyclophosphamide, (EC)        cyclophosphamide/epirubicin/5-fluorouracil (CEF),        cyclophosphamide/doxorubicin/5-fluorouracil (CAF),        5-fluorouracil/epirubicin/cyclophosphamide (FEC)),        cyclophosphamide/metothrexate/5-fluorouracil (CMF),        anthracyclines/taxanes (such as doxorubicin/paclitaxel or        doxorubicin/docetaxel), Docetaxel/capecitabine,        Gemcitabine/paclitaxel, Taxane/platinum regimens (such as        paclitaxel/carboplatin or docetaxel/carboplatin).    -   Hormone therapy is based on the fact that some hormones promote        cancer growth. For example, estrogen in women produced by the        ovaries sometimes promotes the breast cancer growth. There are        several ways for stopping the production of these hormones. A        way is to remove the organs producing them: the ovaries in the        case of women, the testicles in the case of the men. More        frequently, medicaments to prevent these organs from producing        the hormones or to prevent the hormones from acting on the        cancer cells can be used.    -   Immunotherapy is a treatment that aids the immune system itself        of the patient to combat cancer. There are several types of        immunotherapy which are used to treat metastasis patients. These        include but are not limited to cytokines, monoclonal antibodies        and antitumor vaccines.

In another aspect, the treatment is Alpharadin (radium-223 dichloride).Alpharadin uses alpha radiation from radium-223 decay to kill cancercells. Radium-223 naturally self-targets to bone metastases by virtue ofits properties as a calcium-mimic. Alpha radiation has a very shortrange of 2-10 cells (when compared to current radiation therapy which isbased on beta or gamma radiation), and therefore, causes less damage tosurrounding healthy tissues (particularly bone marrow). With similarproperties to calcium, radium-223 is drawn to places where calcium isused to build bone in the body, including the site of faster, abnormalbone growth—such as that seen in the skeletal metastases of men withadvanced, castration-resistant prostate cancer. Radium-223, afterinjection, is carried in the bloodstream to sites of abnormal bonegrowth. The place where a cancer starts in the body is known as theprimary tumor. Some of these cells may break away and be carried in thebloodstream to another part of the body. The canter cells may thensettle in that part of the body and form a new tumor. If this happens itis called a secondary cancer or a metastasis. Most patients with latestage prostate cancer suffer the maximum burden of disease in theirbones. The aim with radium-223 is to selectively target this secondarycancer. Any radium-223 not taken-up in the bones is quickly routed tothe gut and excreted.

In another aspect, the treatment is an mTor inhibitor. In some aspects,the mTor inhibitor is a dual mTor/PI3kinase inhibitor. In some aspects,the mTor inhibitor is used to prevent or inhibit metastasis. In someaspects the mTor inhibitor is selected from the group consisting of:ABI009 (sirolimus), rapamycin (sirolimus), Abraxane (paclitaxel), Absorb(everolimus), Afinitor (everolimus), Afinitor with Gleevec, AS703026(pimasertib), Axxess (umirolimus), AZD2014, BEZ235, Biofreedom(umirolimus), BioMatrix (umirolimus), BioMatrix flex (umirolimus),CC115, CC223, Combo Bio-engineered Sirolimus Eluting Stent ORBUSNEICH(sirolimus), Curaxin CBLC102 (mepacrine), DE109 (sirolimus), DS3078,Endeavor DES (zotarolimus), Endeavor Resolute (zotarolimus), Femara(letrozole), Hocena (antroquinoriol), INK128 Inspiron (sirolimus),IPI504 (retaspimycin hydrochloride), KRN951 (tivozanib), ME344, MGA031(teplizumab), MiStent SES (sirolimus), MKC1, Nobori (umirolimus),OSI027, OVI123 (cordycepin), Palomid 529, PF04691502, Promus Element(everolimus), PWT33597, Rapamune (sirolimus), Resolute DES(zotarolimus), RG7422, SAR245409, SF1126, SGN75 (vorsetuzumabmafodotin), Synergy (everolimus), Taltorvic (ridaforolimus), Tarceva(erlotinib), Torisel (temsirolimus), Xience Prime (everolimus), Xience V(everolimus), Zomaxx (zotarolimus), Zortress (everolimus), ZotarolimusEluting Peripheral Stent MEDTRONIC (zotarolimus), AP23841, AP24170,ARmTOR26, BN107, BN108, Canstatin GENZYME (canstatin), CU906, EC0371,EC0565, KI1004, LOR220, NV128, Rapamycin ONCOIMMUNE (sirolimus), SB2602,Sirolimus PNP SAMYANG BIOPHARMACEUTICALS (sirolimus), TOP216, VLI27,VS5584, WYE125132, XL388, Advacan (everolimus); AZD8055, Cypher SelectPlus Sirolimus eluting Coronary Stent (sirolimuis), Cypher Sirolimuseluting coronary stent (sirolimus), Drug Coated Balloon (sirolimus),E-Magic Plus (sirolimus), Emtor (sirolimus), Esprit (everolimus),Evertor (everolimus), HBF0079, LCP-Siro (sirolimus), Limus CLARIS(sirolimus), mTOR Inhibitor CELLZOME, Nevo Sirolimus eluting CoronaryStent (sirolimus), nPT-mTOR, Rapacan (sirolimus), Renacept (sirolimus),ReZolve (sirolimus), Rocas (sirolimus), SF1126, Sirolim (sirolimus),Sirolimus NORTH CHINA (sirolimus), Sirolimus RANBAXY (sirolimus),Sirolimus WATSON (sirolimus) Siropan (sirolimus), Sirova (sirolimus),Supralimus (sirolimus), Supralimus-Core (sirolimus), Tacrolimus WATSON(tacrolimus), TAFA93; Temsirolimus ACCORD (temsirolimus), TemsirolimusSANDOZ (temsirolimus), TOP216, Xience Prime (everolimus), Xience V(everolimus). In a specific aspect the mTor inhibitor is Afinitor(everolimus)(http://www.afinitor.com/index.jsp?usertrack.filter_applied=true&NovaID=4029462064338207963; last accessed Nov. 28, 2012). In anther aspect, everolimus iscombined with an aromatase inhibitor. (See. e.g., Baselga, J., el al.,Everolimus in Postmenopausal Hormone-Receptor Positive Advanced BreastCancer. 2012. N. Engl. J. Med. 366(6): 520-529, which is hereinincorporated by reference). In another aspect, mTor inhibitors can beidentified through methods known in the art. (See, e.g., Zhou, H. etal., Updates of mTor inhibitors. 2010. Anticancer Agents Med. Chem.10(7): 571-81, which is herein incorporated by reference). In someaspects, the mTor inhibitor is used to treat or prevent or inhibitmetastasis in a patient with advanced lung cancer. In some aspects, themTor inhibitor is used in combination with a second treatment. In someaspects, the second treatment is any treatment described herein.

In another aspect, the treatment is a Src kinase inhibitor. In someaspects, the Src inhibitor is used to prevent or inhibit metastasis: Insome aspects, the Src kinase inhibitor is selected from the group:AZD0530 (saracatinib), Bosulif (bosutinib), ENMD981693, KD020, KX01,Sprycel (dasatinib), Yervoy (ipilimumab), AP23464, AP23485, AP23588,AZD0424, c-Src Kinase Inhibitor KISSEI, CU201, KX2361, SKS927, SRN004,SUNK706, TG100435, TG100948, AP23451, Dasatinib HETERO (dasatinib),Dasatinib VALEANT (dasatinib), Fontrax (dasatinib), Src Kinase InhibitorKINEX, VX680, (tozasertib lactate), XL228, and SUNK706. In someembodiments, the Src kinase inhibitor is dasatinib. In another aspect,Src kinase inhibitors can be identified through methods known in the art(See, e.g., Sen, B. and Johnson, F. M. Regulation of Src Family Kinasesin Human Cancers. 2011. J. Signal Transduction. 2011: 14 pages, which isherein incorporated by reference). In some aspects, the Src kinaseinhibitor is used to treat or prevent or inhibit metastasis in a patientthat is positive for the SRC-responsive signature (SRS). In someaspects, the patient is SRS+ and ER−. (See. e.g., Zhang, C H-F, et al.Latent Bone Metastasis in Breast Cancer Tied to Src-Dependent survivalsignals. 2009. Cancer Cell. 16:67-78, which is herein incorporated byreference.) In some aspects, the Src kinase inhibitor is used to treator prevent or inhibit metastasis in a patient with advanced lung cancer.In some aspects, the Src kinase inhibitor is used in combination with asecond treatment. In some aspects, the second treatment is any treatmentdescribed herein.

In another aspect, the treatment is a COX-2 inhibitor. In some aspects,the COX-2 inhibitor is used to prevent or inhibit metastasis. In someaspects, the COX-2 inhibitor is selected from the group: ABT963,Acetaminophen ER JOHNSON (acetaminophen); Acular X (ketorolactromethamine), BAY1019036 (aspirin), BAY987111 (diphenhydramine,naproxen sodium), BAY11902 (piroxicam), BCIBUCH001 (ibuprofen),Capoxigem (apricoxib), CS502, CS670 (pelubiprofen), Diclofenac HPBCD(diclofenac), Diractin (ketoprofen), GW406381, HCT1026(nitroilurbiprofen), Hyanalgese-D (diclofenac), HydrocoDex(acetaminophen, dextromethorphan, hydrocodone), Ibuprofen Sodium PFIZER(ibuprofen sodium), Ibuprofen with Acetaminophen PFIZER (acetaminophen,ibuprofen), Impracor (ketoprofen), IP880 (diclofenac); IP940(indomethacin), ISV205 (diclofenac Sodium), JNS013 (acetaminophen,tramadol hydrochloride), Ketoprofen TDS (ketoprofen), LTNS001 (naproxenetemesil), Mesalamine SALIX (mesalamine), Mesalamine SOFAR (mesalamine),Mesalazine (mesalamine), ML3000 (licofelone), MRX7EAT (etodolac),Naproxen IROKO (naproxen), NCX4016 (nitroaspirin), NCX701(nitroacetaminophen), Nuprin SCOLR (ibuprofen), OMS103HP (amitriptylinehydrochloride, ketoprofen, oxymetazoline hydrochloride), Oralease(diclofenac), OxycoDex (dextromethorphan, oxycodone), P54, PercoDex(acetaminophen, dextromethorphan, oxycodone), PL3100 (naproxen,phosphatidyl choline), PSD508, R-Ketoprofen (ketoprofen), Remura(bromfenac sodium), ROX828 (ketorolac tromethamine), RP19583 (ketoprofenlysine), RQ00317076, SDX101 (R-etodolac), TDS943 (diclofenac sodium),TDT070 (ketoprofen), TPR100, TQ1011 (ketoprofen), TT063(S-flurbiprofen), UR8880 (cimicoxib), V0498TA01A (ibuprofen), VT122(etodolac, propranolol), XP20B (acetaminophen, dextropropoxyphene),XP21B (diclofenac potassium), XP21L (diclofenac potassium), Zoenasa(acetylcysteine, mesalamine), Acephen, Actifed Plus, Actifed-P, Acular,Acular LS, Acular PF, Acular X, Acuvail, Advil, Advil Allergy Sinus,Advil Cold and Sinus, Advil Congestion Relief, Advil PM, Advil PMCapsule, Air Salonpas, Airtal, Alcohol-Free NyQuil Cold & Flu Relief,Aleve, Aleve ABDI IBRAHIM, Aleve-D, Alka-Seltzer, Alka-Seltzer BAYER,Alka-Seltzer Extra Strength, Alka-Seltzer Lemon-Lime, Alka-SeltzerOriginal, Alka-Seltzer Plus, Alka-Seltzer plus Cold and Cough,Alka-Seltzer plus Cold and Cough Formula, Alka-Seltzer Plus Day andNight Cold Formula, Alka-Seltzer Plus Day Non-Drowsy Cold Formula,Alka-Seltzer Plus Flu Formula, Alka-Seltzer Plus Night Cold Formula,Alka-Seltzer Plus Sinus Formula, Alka-Seltzer Plus Sparkling OriginalCold Formula, Alka-Seltzer PM, Alka-Seltzer Wake-Up Call, Anacin,Anaprox, Anaprox MINERVA, Ansaid, Apitoxin, Apranak, Apranax abdi,Arcoxia, Arthritis Formula Bengay, Arthrotec, Asacol, Asacol HD, AsacolMEDUNA ARZNEIMITTEL, Asacol ORIFARM, Aspirin BAYER, Aspirin Complex,Aspirin Migran, AZD3582, Azulfidine, Baralgan M, BAY1019036, BAY987111,BAY11902, BCIBUCH001, Benadryl Allergy, Benadryl Day and Night, Benylin4 Flu, Benylin Cold and Flu, Benylin Cold and Flu Day and Night, BenylinCold and Sinus Day and Night, Benylin Cold and Sinus Plus, Benylin Dayand Night Cold and Flu Relief, Benylin1 All-In-One, Brexin, BrexinANGELINI, Bromday, Bufferin, Buscopan Plus, Caldolor, Calmatel, Cambia,Canasa, Capoxigem, Cataflam, Celebrex, Celebrex ORIFARM, Children'sAdvil Allergy Sinus, Children's Tylenol, Children's Tylenol Cough andRunny Nose, Children's Tylenol plus cold, Children's Tylenol plus Coldand Cough, Children's Tylenol plus cold and stuffy nose, Children'sTylenol plus Flu, Children's Tylenol plus cold & allergy, Children'sTylenol plus Cough & Runny Nose, Children's Tylenol plus Cough & SoreThroat, Children's Tylenol plus multi symptom cold, Clinoril, CodralCold and Flu, Codral Day and Night Day Tablets, Codral Day and NightNight Tablets, Codral Nightime, Colazal, Combunox, Contac Cold plus Flu,Contac Cold plus Flu Non-Drowsy, Coricidin D, Coricidin HBP Cold andFlu, Coricidin HBP Day and Night Multi-Symptom Cold, Coricidin HBPMaximum Strength Flu, Coricidin HBP Nighttime Multi-Symptom Cold,Coricidin II Extra Strength Cold and Flu, CS502, CS670, Daypro, DayproAlta, DDS06C, Demazin Cold and Flu, Demazin Cough, Cold and Flu, Demazinday/night Cold and Flu, Demazin PE Cold and Flu, Demazin PE day/nightCold and Flu, Diclofenac HPBCD, Dimetapp Day Relief, DimetappMulti-Symptom Cold and Flu, Dimetapp Night Relief, Dimetapp Pain andFever Relief, Dimetapp PE Sinus Pain, Dimetapp PE Sinus Pain plusAllergy, Dipentum, Diractin, Disprin Cold 'n' Fever, Disprin Extra,Disprin Forte. Disprin Plus, Dristan Cold, Dristan Junior, DrixoralPlus, Duexis, Dynastat, Efferalgan, Efferalgan Plus Vitamin C,Efferalgan Vitamin C, Elixsure IB, Excedrin Back and Body, ExcedrinMigraine, Excedrin PM, Excedrin Sinus Headache, Excedrin TensionHeadache, Falcol, Fansamac, Feldene, FeverAll, Fiorinal, Fiorinal withCodeine, Flanax, Flector Patch, Flucam, Fortagesic, Gerbin, Giazo,Gladio, Goody's Back and Body Pain, Goody's Cool Orange, Goody's ExtraStrength, Goody's PM, Greaseless Bengay, GW406381, HCT1026, He Xing Yi,Hyanalgese-D, HydrocoDex, Ibuprofen Sodium PFIZER, Ibuprofen with,Acetaminophen PFIZER, Icy Hot SANOFI AVENTIS, Impracor, Indocin,Indomethacin APP PHARMA, Indomethacin MYLAN, Infants' Tylenol, IP880,IP940, Iremod, ISV205, JNS013, Jr. Tylenol, Junifen, Junior StrengthAdvil, Junior Strength Motrin, Ketoprofen TDS, Lemsip Max, Lemsip MaxAll in One, Lemsip Max All Night, Lemsip Max Cold and Flu, Lialda,Listerine Mouth Wash, Lloyds Cream, Lodine, Lorfit P, Loxonin, LTNS001,Mersyndol, Mesalamine SALIX, Mesalamine SOFAR, Mesalazine, MesasalGLAXO, Mesasal SANOFI, Mesulid, Metsal Heat Rub, Midol Complete, MidolExtended Relief, Midol Liquid Gels, Midol PM, Midol Teen Formula,Migranin COATED TABLETS, ML3000, Mobic, Mohrus, Motrin, Motrin Cold andSinus Pain, Motrin PM, Movalis ASPEN, MRX7EAT, Nalfon, Nalfon PEDTNOL,Naprelan, Naprosyn, Naprosyn RPG LIFE SCIENCE, Naproxen IROKO, NCX4016,NCX701, NeoProfen LUNDBECK, Nevanac, Nexcede, Niflan, Norgesic MEDICIS,Novalgin, Nuprin SCOLR, Nurofen, Nurofen Cold and Flu, Nurofen MaxStrength Migraine, Nurofen Plus, Nuromol, NyQuil with Vitamin C, Ocufen,GMS103HP, Oralease, Orudis ABBOTT JAPAN, Oruvail, Osteluc, OxycoDex,P54, Panadol, Panadol Actifast, Paradine, Paramax, Parfenac, Pedea,Pennsaid, Pentasa, Pentasa ORIFARM, Peon, Percodan, Percodan-Demi,PercoDex, Percogesic, Perfalgan, PL2200, PL3100, Ponstel, Prexige,Prolensa, PSD508, R-Ketoprofen, Rantudil, Relafen, Remura, Riobaxisal,Rotec, Rowasa, ROX828, RP19583, RQ00317076, Rubor, Salofalk, Salonpas,Saridon, SDX101, Seltouch, sfRowasa, Shinbaro, Sinumax, Sinutab,Sinutab, sinus, Spalt, Sprix, Strefen, Sudafed Cold and Cough, SudafedHead Cold and Sinus, Sudafed PE Cold plus Cough, Sudafed PE Pressureplus Pain, Sudafed PE, Severe Cold, Sudafed PE Sinus Day plus NightRelief Day Tablets, Sudafed PE Sinus Day plus Night Relief NightTablets, Sudafed PE Sinus plus Anti-inflammatory Pain Relief, SudafedSinus Advance, Surgam, Synalgos-DC, Synflex, Tavistallergy/sinus/headache, TDS943, TDT070, Theraflu Cold and Sore Throat,Theraflu Daytime Severe Cold and Cough, Theraflu Daytime Warming Relief,Theraflu Warming Relief Caplets Daytime Multi-Symptom Cold, TherafluWarming Relief Cold and Chest Congestion, Thomapyrin, Thomapyrin C,Thomapyrin Effervescent, Thomapyrin Medium, Tilcotil, Tispol, Tolectin,Toradol, TPR100, TQ1011, Trauma-Salbe, Trauma-Salbe Kwizda, Treo,Treximet, Trovex, TT063, Tylenol, Tylenol Allergy Multi-Symptom; TylenolBack Pain, Tylenol Cold & Cough Daytime, Tylenol Cold & Cough Nighttime,Tylenol Cold and Sinus Daytime, Tylenol Cold and Sinus Nighttime,Tylenol Cold Head Congestion Severe, Tylenol Cold Multi Symptom Daytime,Tylenol Cold Multi Symptom Nighttime Liquid, Tylenol Cold Multi SymptomSevere, Tylenol Cold Non-Drowsiness Formula, Tylenol Cold SevereCongestion Daytime, Tylenol Complete Cold, Cough and Flu Night time,Tylenol Flu Nighttime, Tylenol Menstrual, Tylenol PM, Tylenol SinusCongestion & Pain Daytime, Tylenol Sinus Congestion & Pain Nighttime,Tylenol Sinus Congestion & Pain Severe, Tylenol Sinus Severe CongestionDaytime, Tylenol Ultra Relief, Tylenol with Caffeine and Codeinephosphate, Tylenol with Codeine phosphate, Ultra Strength Bengay Cream,Ultracet, UR8880, V0498TA01A, Vicks NyQuil Cold and Flu Relief,Vicoprofen, Vimovo, Voltaren Emulgel, Voltaren GEL, Voltaren NOVARTISCONSUMER HEALTH GMBH, Voltaren XR, VT122, Xefo, Xefo Rapid, Xefocam,Xibrom, XL3, Xodol, XP20B, XP21B, XP21L, Zipsor, and Zoenasa. In anotheraspect, COX-2 inhibitors can be identified through methods known in theart (See, e.g., Dannhardt, G. and Kiefer, W. Cyclooxygenaseinhibitors—current status and future prospects. 2001. Eur. J. Med. Chem.36:109-126, which is herein incorporated by reference). In some aspects,the COX-2 inhibitor is used to treat or prevent or inhibit metastasis ina patient with advanced lung cancer. In some aspects, the COX-2inhibitor is used in combination with a second treatment. In someaspects, the second treatment is any treatment described herein. In someaspects, the COX-2 inhibitor is used in combination with a secondtreatment selected from the group consisting of: Denosumab, Zometa(http://www.us.zometa.com/index.jsp♭usertrack.filter_applied=true&NovaId=2935376934467633633; last accessed Dec. 2, 2012), Carbozantinib or Cabozantinib,Antibody or peptide blocking PTHLH (parathyroid hormone like hormone) orPTHrP (parathyroid hormone related protein).

In another aspect, the treatment agents used for avoiding and/orpreventing bone degradation include, but are not limited to:

-   -   Parathyroid hormone (PTH) and Parathyroid like hormone (PTHLH)        inhibitors (including blocking antibodies) or recombinant forms        thereof (teriparatide corresponding to the amino acids 7-34 of        PTH). This hormone acts by stimulating the osteoclasts, and        increasing their activity.    -   Strontium ranelate: is an alternative oral treatment, and forms        part of the group of drugs called “dual action bone agents”        (DABAs) because they stimulate the osteoblast proliferation and        inhibit the osteoclast proliferation.    -   “Estrogen receptor modulators” (SERM) refers to compounds which        interfere or inhibit the binding of estrogens to the receptor,        regardless of the mechanism. Examples of estrogen receptor        modulators include, among others, estrogens progestagen,        estradiol, droloxifene, raloxifene, lasofoxifene, TSE-424,        tamoxifen, idoxifene, L Y353381, LY117081, toremifene,        fluvestrant, 4-[7-(2,2-dimethyl-1-oxopropoxy        -4-methyl-2-[4-[2-(1-piperidinyl)ethoxy]phenyl]-2H-1-benzopyran-3-yl]-phenyl-2,2-dimethylpropanoate        4,4′dihydroxybenzophenone-2,4-dinitrophenyl-hydrazone and SH646.        -   Calcitonin: directly inhibits the osteoclast activity            through the calcitonin receptor. The calcitonin receptors            have been identified on the surface of the osteoclasts.    -   Bisphosphonates: are a group of medicinal products used for the        prevention and the treatment of diseases with bone resorption        and reabsorption such as osteoporosis and cancer with bone        metastasis, the latter being with or without hypercalcaemia,        associated to breast cancer and prostate cancer. Examples of        bisphosphonates which can be used in the therapy designed by        means of the fifth method of the invention, include, although        not limited to, nitrogenous bisphosphonates (such as        pamidronate, neridronate, olpadronate, alendronate, ibandronate,        risedronate, incadronate, zoledronate or zoledronic acid etc.)        and non-nitrogenous bisphosphonates (such as etidronate,        clodronate, tiludrdnate, etc.).    -   “Cathepsin K inhibitors” refers to compounds which interfere in        the cathepsin K cysteine protease activity. Non-limiting        examples of cathepsin K inhibitors include        4-amino-pyrimidine-2-carbonitrile derivatives (described in the        International patent application WO 03/020278 under the name of        Novartis Pharma GMBH), pyrrolo-pyrimidines described in the        publication WO 03/020721 (Novartis Pharma GMBH) and the        publication WO 04/000843 (ASTRAZENECA AB) as well as the        inhibitors described in the publications PCT WO 00/55126 of Axys        Pharmaceuticals, WO 01/49288 of Merck Frosst Canada & Co. and        Axys Pharmaceuticals.    -   “DKK-1 (Dickkopf-1) inhibitor” as used herein refers to any        compound which is capable of reducing DKK-1 activity. DKK-1 is a        soluble Wnt pathway antagonist expressed predominantly in adult        bone and upregulated in myeloma patients with osteolytic        lesions. Agents targeting DKK-1 may play a role in preventing        osteolytic bone disease in multiple myeloma patients. BHQ880        from Novartis is a first-in-class, fully human, anti-DKK-1        neutralizing antibody. Preclinical studies support the        hypothesis that BHQ880 promotes bone formation and thereby        inhibits tumor-induced osteolytic disease (Ettenberg S. et al.,        American Association for Cancer Research Annual Meeting, Apr.        12-16, 2008; San Diego, Calif. Abstract).    -   “Dual MET and VEGFR2 inhibitor” as used herein refers to any        compound which is a potent dual inhibitor of the MET and VEGF        pathways designed to block MET driven tumor escape. MET is        expressed not only in tumor cells and endothelial cells, but        also in osteoblasts (bone-forming cells) and osteoclasts        (bone-removing cells). HGF binds to MET on all of these cell        types, giving the MET pathway an important role in multiple        autocrine and paracrine loops. Activation of MET in tumor cells        appears to be important in the establishment of metastatic bone        lesions. At the same time, activation of the MET pathway in        osteoblasts and osteoclasts may lead to pathological features of        bone metastases, including abnormal bone growth (ie, blastic        lesions) or destruction (ie, lytic lesion. Thus, targeting the        MET pathway may be a viable strategy in preventing the        establishment and progression of metastatic bone lesions.        Cabozantinib (Exelixis, Inc), formerly, known as XL184 (GAS        849217-68-1), is a potent dual inhibitor of the MET and VEGF        pathways designed to block MET driven tumor escape. In multiple        preclinical studies cabozantinib has been shown to kill tumor        cells, reduce metastases, and inhibit angiogenesis (the        formation of new blood vessels necessary to support tumor        growth). Another suitable dual inhibitors are E7050        (N-[2-Fluoro-4-({2-[4-(4-methylpiperazin-1-yl)piperidin-1-yl]carbonylaminopyridin-4-}        oxy) phenyl]-N′-(4-fluorophenyl) cyclopropane-1,1-dicarboxamide        (2R,3R)-tartrate) (GAS 928037-13-2) or Foretinib (also known as        GSK1363089, XO880, CAS 849217-64-7).    -   “RANKL inhibitors” as used herein refer to any compound which is        capable of reducing the RANK activity. RANKL is found on the        surface of the osteoblast membrane of the stroma and        T-lymphocyte cells; and these T-lymphocyte cells are the only        ones which have demonstrated the capacity for secreting it. Its        main function is the activation of the osteoclasts cells        involved in the bone resorption. The RANKL inhibitors can act by        blocking the binding of RANKL to its receptor (RANK), blocking        the RANK-mediated signaling or reducing the expression of RANKL        by blocking the transcription or the translation of RANKL. RANKL        antagonists or inhibitors suitable for use in the present        invention include, without limitation:        -   a suitable RANK protein which is capable of binding RANKL            and which comprises the entire or a fragment of the            extracellular domain of a RANK protein. The soluble RANK,            may comprise the signal peptide and the extracellular domain            of the murine or human RANK polypeptides, or alternatively,            the mature form of the protein with the signal peptide            removed can be used.        -   Osteoprotegerin or a variant thereof with RANKL-binding            capacity.        -   RANKL-specific antisense molecules        -   Ribozymes capable of processing the transcribed products of            RANKL        -   Specific anti-RANKL antibodies. “Anti-RANKL antibody or            antibody directed against RANKL” is understood herein as all            that antibody which is capable of binding specifically to            the ligand of the activating receptor for the nuclear factor            κB (RANKL) inhibiting one or more RANKL functions. The            antibodies can be prepared using any of the methods which            are known by the person skilled in the art. Thus, the            polyclonal antibodies are prepared by means of immunizing an            animal with the protein to be inhibited. The monoclonal            antibodies are prepared using the method described, by            Kohler, Milstein et al (Nature, 1975, 256: 495). Antibodies            suitable in the context of the present invention include            intact antibodies which comprise a variable, antigen binding            region and a constant region, fragments “Fab”, “F(ab′)2” and            “Fab”, Fv, scFv, diabodies and bispecific antibodies.        -   Specific anti-RANKL nanobodies. Nanobodies are            antibody-derived therapeutic proteins that contain the            unique structural and functional properties of            naturally-occurring heavy-chain antibodies. The Nanobody            technology was originally developed following the discovery            that camelidae (camels and llamas) possess fully functional            antibodies that lack light chains. The general structure Of            nanobodies is

FR1-CDR1-FR2-CDR2-FR3-CPR3-FR4

wherein FR1 to FR4 are the framework regions 1 to 4 CDR1 to CDR3 are thecomplementarity determining regions 1 to 3. These heavy-chain antibodiescontain a single variable domain (VHH) and two constant domains (CH2 andCH3). Importantly, the cloned and isolated VHH domain is a perfectlystable polypeptide harbouring the full antigen-binding capacity of theoriginal heavy-chain antibody. These newly discovered VHH; domains withtheir unique structural and functional properties form the basis of anew generation of therapeutic antibodies which Ablynx has namedNanobodies.

In one embodiment, the RANKL inhibitor is selected from the groupconsisting of a RANKL specific antibody, a RANKL specific nanobody andosteoprotegerin. In a specific embodiment, the anti-RANKL antibody is amonoclonal antibody. In a yet more specific embodiment, the anti-RANKLantibody, is Denosumab (Pageau, Steven C. (2009). mAbs 1 (3): 210-215,CAS number 615258-40-7) (the entire contents of which are herebyincorporated by reference). Denosumab is a fully human monoclonalantibody which binds to RANKL and prevents its activation (it does notbind to the RANK receptor). Various aspects of Denosumab are covered byU.S. Pat. Nos. 6,740,522; 7,411,050; 7,097,834; 7;364,736 (the entirecontents of each of which are hereby incorporated by reference in theirentirety). In another embodiment, the RANKL inhibitor an antibody,antibody fragment, or fusion construct that binds the same epitope asDenosumab.

In a preferred embodiment; the anti-RANKL nanobody is any of thenanobodies as described in WO2008142164, (the contents of which areincorporated in the present application by reference). In a still, morepreferred embodiment, the anti-RANKL antibody is the ALX-0141 (Ablynx).ALX-0141 has been designed to inhibit bone loss associated withpost-menopausal osteoporosis, reumatoid arthritis, cancer and certainmedications, and to restore the balance of healthy bone metabolism.

In a preferred embodiment, the agent preventing the bone degradation isselected from the group consisting of a bisphosphonate, a RANKLinhibitor, PTH and PTHLH inhibitor or a PRG analog, strontium ranelate,a DKK-1 inhibitor, a dual MET and VEGFR2inhibitor, Radium-223calcitonin, and a cathepsin K inhibitor. In a more preferred embodimentthe agent preventing the bone degradation is a bisphosphonate. In a yetmore preferred embodiment, the bisphosphonate is the zoledronic acid.

In one embodiment, a CCR5 antagonist is administered to prevent orinhibit metastasis of the primary lung cancer tumor to bone. In oneembodiment, the CCR5 antagonist is a large molecule. In anotherembodiment, the CCR5 antagonist is a small molecule. In someembodiments, the CCR5 antagonist is Maraviroc (Velasco-Veláquez, M. etal. 2012. CCR5 Antagonist Blocks Metastasis of Basal Breast CancerCells. Cancer Research, 72:3839-3850.) In some embodiments, the CCR5antagonist is Vicriviroc. Velasco-Veláquez, M. et al 2012. CCR5Antagonist Blocks Metastasis of Basal Breast Cancer Cells. CancerResearch. 72:3839-3850.). In some aspects, the CCR5 antagonist isAplaviroc (Demarest J. F. et al. 2005. Update on Aplaviroc: An HIV EntryInhibitor Targeting CCR5. Retrovirology 2(Suppl. 1): S13). In someaspects, the CCR5 antagonist is a spiropiperidine CCR5 antagonist.(Rotstein D. M. et al. 2009. Spiropiperidine CCR5 antagonists.Bioorganic & Medicinal Chemistry Letters. 19(18): 5401-5406. In someembodiments, the CCR5 antagonist is INCB009471 (Kuritzkes, D. R. 2009.HIV-1 entry inhibitors: an overview. Curr. Opin. HIV AIDS. 4(2): 82-7).

In a preferred embodiment the dual MET and VEGFR2 inhibitor is selectedfrom the group consisting of Cabozantinib, Foretinib and E7050.

In a preferred embodiment the Radium 223 therapy is alpharadin.

Alternatively a combined treatment can be carried out in which more thanone agent from those mentioned above are combined to treat and/orprevent the metastasis or said agents can be combined with othersupplements, such as calcium or vitamin D or with a hormone treatment.

Method for Designing Customized Therapy of the Invention in Lung CancerPatients with Bone Metastasis

Patients suffering lung cancer which has already metastasized to thebone and in which there are elevated c-MAF levels may particularlybenefit from therapies aimed at preventing the bone degradation causedby the increased osteoclastic activity.

Thus, in another aspect, the invention relates to an in vitro method fordesigning a customized therapy for a subject with lung cancer with bonemetastasis (hereinafter third method of the invention) which comprises

-   -   (i) quantifying the c-MAF gene expression level in a metastatic        tumor sample from bone of said subject, and    -   (ii) comparing the expression/level previously obtained with the        expression level of said gene in a control sample,        wherein if the expression levels are increased with respect to        the expression levels of said gene in the control sample, then        said subject is susceptible to receive a therapy aiming to        prevent the bone degradation.        wherein if the expression level is not increased with respect to        said reference value, then said subject is not susceptible to        receive a therapy aiming to prevent and/or treat the bone        metastasis.

The terms and expressions “subject”, “lung cancer”, “tumor sample”,“metastasis”, “determination of expression levels”, “c-MAF gene”,“increased expression levels” and “control sample” have been describedin detail in relation to the first method of the invention and areequally applicable to the second and third method of the invention.

In a preferred embodiment, the bone metastasis is osteolytic metastasis.

The third method of the invention comprises in a first step, quantifyingthe c-MAF gene expression level in a tumor sample in a subject sufferinglung cancer. In the case of the third method of the invention, thesample is a tissue sample from bone metastasis.

In a preferred embodiment, the third method of the invention comprisesquantifying only the c-MAF gene expression level as a single marker,i.e., the method does not involve determining the expression level ofany additional marker.

In a second step the c-MAF gene expression level obtained in the tumorsample of the subject is compared with the expression level of said genein a control sample. The determination of the c-MAF gene expressionlevels must be correlated to values of a control sample or referencesample. Depending on the type of tumor to be analyzed, the exact natureof the control sample may vary. Thus, in the case involving the thirdmethod of the invention, then the reference sample is a tumor tissuesample of subject with lung cancer who has not suffered metastasis orthat correspond to the median value of the c-MAF gene expression levelsmeasured in a tumor tissue collection in biopsy samples of subjects withlung cancer who has not suffered metastasis.

Once the c-MAF gene expression level in the sample is measured andcompared with the control sample, if the expression level of said geneare increased with respect to its expression levels in the controlsample, then it can be concluded that said subject is susceptible toreceive a therapy aiming to avoid or prevent bone degradation.

As used herein, an “agent for avoiding or preventing bone degradation”refers to any molecule capable of treating or stopping bone degradationeither by stimulating the osteoblast proliferation or inhibiting theosteoclast proliferation.

Illustrative examples of agents used for avoiding and/or preventing bonedegradation and/or bone metastasis include, although not limited to:

-   -   Parathyroid hormone (PTH) or recombinant forms thereof        (teriparatide corresponding to the amino acids 1-34 of PTH).        This hormone acts by stimulating the osteoblasts and increasing        their activity.    -   Strontium ranelate: is an alternative oral treatment, and forms        part of the group of drugs called “dual action/bone agents”        (DABAs) because they stimulate the osteoblast proliferation and        inhibit the osteoclast proliferation.    -   “Estrogen receptor modulators” (SERM) refers to compounds which        interfere or inhibit the binding of estrogens to the receptor,        regardless of the mechanism. Examples of estrogen receptor        modulators include, among others, estrogens progestagen,        estradiol, droloxifene, raloxifene, lasofoxifene, TSE-424,        tamoxifen, idoxifene, L Y353381, LY117081, toremifene,        fluvestrant, 4-[7-(2,2-dimethyl-1-oxopropoxy        -4-methyl-2-[4-[2-(1-piperidinyl)ethoxy]phenyl]-2H-1-benzopyran-3-yl]-phenyl-2,2-dimethylpropanoate        4,4′dihydroxybenzophenone-2,4-dinitrophenyl-hydrazone and SH646.    -   Calcitonin: directly inhibits the osteoclast activity through        the calcitonin receptor. The calcitonin receptors have been        identified, on the surface of the osteoclasts.    -   Bisphosphonates: are a group of medicinal products used for the        prevention and the treatment of diseases with bone resorption        and reabsorption such as osteoporosis and cancer with bone        metastasis, the latter being with or without hypercalcaemia;        associated to breast cancer and prostate cancer. Examples of        bisphosphonates which cane be used in the therapy designed by        means of the third method of the invention include, although not        limited to, nitrogenous bisphosphonates (such as pamidronate,        neridronate, olpadronate, alendronate, ibandronate, risedronate,        incadronate, zoledronate or zoledronic acid, etc.) and        non-nitrogenous bisphosphonates (such as etidronate, clodronate,        tiludronate, etc.).    -   “Cathepsin K inhibitors” refers to compounds which interfere in        the cathepsin K cysteine protease activity. Non-limiting        examples of cathepsin K inhibitors include        4-amino-pyrimidine-2-carbonitrile derivatives (described in the        International patent application WO 03/020278 under the name of        Novartis Pharma GMBH), pyrrolo-pyrimidines described in the        publication WO 03/020721 (Novartis Pharma GMBH) and the        publication WO 04/000843 (ASTRAZENECA AB) as well as the        inhibitors described/in the publications PCT WO 00/55126 of Axys        Pharmaceuticals, WO 01/49288 of Merck Frosst Canada & Co. and        Axys Pharmaceuticals.    -   “DKK-1 (Dickkopf-1) inhibitor” as used herein refers to any        compound which is capable of reducing DKK-1 activity. DKK-1 is a        soluble Wnt pathway antagonist expressed predominantly in adult        bone and upregulated in myeloma patients with osteolytic        lesions. Agents targeting DKK-1 may play a role in preventing        osteolytic bone disease in multiple myeloma patients. BHQ880        from Novartis is a first-in-class, fully human,        anti-DKK-1neutralizing antibody. Preclinical studies support the        hypothesis that BHQ880 promotes bone formation and thereby        inhibits tumor-induced osteolytic disease. (Ettenberg S. et al.,        American Association for Cancer Research Annual Meeting. Apr.        12-16, 2008; San Diego, Calif. Abstract).    -   “Dual MET and VEGFR2 inhibitor” as used herein refers to any        compound which is a potent dual inhibitor of the MET and VEGF        pathways designed to block MET driven tumor escape. MET is        expressed not only in tumor cells and endothelial cells, but        also, in osteoblasts (bone-forming cells) and osteoclasts        (bone-removing cells). HGF binds to MET on all of these cell        types, giving the MET pathway an important role in multiple        autocrine and paracrine loops. Activation of MET in tumor cells        appears to be important in the establishment of metastatic bone        lesions. At the same time, activation of the MET pathway in        osteoblasts and osteoclasts may lead to pathological features of        bone metastases, including abnormal bone growth (ie, blastic        lesions) or destruction (ie, lytic lesion. Thus, targeting the        MET pathway may be a viable strategy in preventing the        establishment and progression of metastatic bone lesions.        Cabozantinib (Exelixis, Inc), formerly known as XL184 (CAS        849217-68-1), is a potent dual inhibitor of the MET and VEGF        pathways designed to block MET driven tumor escape. In multiple        preclinical studies cabozantinib has been shown to kill tumor        cells, reduce metastases, and inhibit angiogenesis (the        formation of new blood vessels necessary to support tumor        growth). Another suitable dual inhibitors are E7050        (N-[2-Fluoro-4-({2-[4-(4-methylpiperazin-1-yl)piperidin-1-yl]carbonylaminopyridin-4-yl}oxy)phenyl]-N′-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide        (2R,3R)-tartrate) (CAS 928037-13-2) or Foretinib (also known as        GSK1363089, XL880, CAS 849217-64-7).    -   “RANKL inhibitors” as used herein refers to any compound which        is capable of reducing the RANK activity. RANKL is found on the        surface of the osteoblast membrane of the stroma and        T-lymphocyte cells, and these T-lymphocyte cells are the only        ones which have demonstrated the capacity for secreting it. Its        main function is the activation of the osteoclasts, cell's        involved in the bone resorption. The RANKL inhibitors can act by        blocking the binding of RANKL to its receptor (RANK), blocking        the RANK-mediated signaling or reducing the expression of RANKL        by blocking the transcription or the translation of RANKL. RANKL        antagonists or inhibitors suitable for use in the present        invention include, without limitation:        -   a suitable RANK protein which is capable of binding RANKL            and which comprises the entire or a fragment of the            extracellular domain of a RANK protein. The soluble RANK may            comprise the signal peptide and the extracellular domain of            the murine or human RANK polypeptides, or alternatively, the            mature form of the protein with the signal peptide removed            can be used.        -   Osteoprotegerin or a variant thereof with RANKL-binding            capacity.        -   RANKL-specific antisense molecules        -   Ribozymes capable of processing the transcribed products of            RANKL        -   Specific anti-RANKL antibodies. “Anti-RANKL antibody or            antibody directed against RANKL” is understood herein as all            that antibody which is capable of binding specifically to            the ligand of the activating receptor for the nuclear factor            κB (RANKL) inhibiting one or more RANKL functions. The            antibodies can be prepared using any of the methods which            are known by the person skilled in the art. Thus, the            polyclonal antibodies are prepared by means of immunizing an            animal with the protein to be inhibited. The monoclonal            antibodies are prepared using the method described by            Kohler, Milstein et al. (Nature, 1975, 256: 495). Antibodies            suitable in the context of the present invention include            intact antibodies which comprises a variable antigen binding            region and a constant region, fragments “Fab”, “F(ab′)2” and            “Fab′”, Fv, scFv, diabodies and bispecific antibodies.        -   Specific anti-RANKL nanobodies. Nanobodies are            antibody-derived therapeutic proteins that contain the            unique structural and functional properties of            naturally-occurring heavy-chain antibodies. The Nanobody            technology was originally developed following the discovery            that camelidae (camels and llamas) possess fully functional            antibodies that lack light chains. The general structure of            nanobodies is

FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 wherein FR1 to FR4 are the frameworkregions 1 to 4 CDR1 to CDR3 are the complementarity determining regions1 to 3. These heavy-chain antibodies contain a single variable domain(VHH) and two constant domains (CH2 and CH3). Importantly, the clonedand isolated VHH domain is a perfectly stable polypeptide harbouring thefull antigen-binding capacity of the original heavy-chain antibody.These newly discovered VHH domains with their unique structural andfunctional propertied form the basis of a new generation of therapeuticantibodies which Ablynx has named Nanobodies.

In one embodiment, the RANKL inhibitor is selected from the groupconsisting of a RANKL specific antibody, a RANKL specific nanobody andosteoprotegerin. In a preferred embodiment, the anti-RANKL antibody is amonoclonal antibody. In a yet more preferred embodiment, the anti-RANKLantibody is Denosumab (Pageau, Steven C. (2009). mAbs 1 (3): 210-215,CAS number 615258-40-7). In the context of the present invention,Denosumab is a monoclonal antibody which binds to RANKL and prevents itsactivation (it does not bind to the RANK receptor). Various aspects ofDenosumab are covered by U.S. Pat. Nos. 6,740,522; 7,411,050; 7,097,834;7,364,736 (the entire contents of each of which are hereby incorporatedby reference in their entirety). In another embodiment, the RANKLinhibitor is an antibody, antibody fragment, or fusion construct thatbinds the same epitope as Denosumab.

In a preferred embodiment, the anti-RANKL nanobody is any of thenanobodies as described in WO2008142164, (the contents of which areincorporated in the present application by reference). In a still morepreferred embodiment, the anti-RANKL antibody is the ALX-0141 (Ablynx).ALX-0141 has been designed to inhibit bone loss associated withpost-menopausal osteoporosis, reumatoid arthritis, cancer and certainmedications, and to restore the balance of healthy bone metabolism.

In a preferred embodiment, the agent preventing the bone degradation isselected from the group consisting of a bisphosphonate, a RANKLinhibitor, PTH and PTHLH inhibitor or a PRG analog, strontium ranelate,a DKK-1 inhibitor, a dual MET and VEGFR2 inhibitor, an estrogen receptormodulator, Radium-223 calcitonin, and a cathepsin K inhibitor. In a morepreferred embodiment the agent preventing the bone degradation is abisphosphonate. In a yet more preferred embodiment, the bisphosphonateis the zoledronic acid.

In one embodiment, a CCR5 antagonist is administered to prevent orinhibit metastasis of the primary lung cancer tumor to bone. In oneembodiment, the CCR5 antagonist is a large molecule. In anotherembodiment, the CCR5 antagonist is a small molecule. In someembodiments, the CCR5 antagonist is Maraviroc (Velasco-Veláquez, M. et.al. 2012. CCR5 Antagonist Blocks Metastasis of Basal Breast CancerCells. Cancer Research. 72:3839-3850.). In some embodiments, the CCR5antagonist is Vicriviroc. Velasco-Veláquez, M. et al. 2012. CCR5Antagonist Blocks Metastasis of Basal Breast Cancer Cells. CancerResearch. 72:3839-3850). In some aspects, the CCR5 antagonist isAplaviroc (Demarest J. F. et al. 2005. Update on Aplaviroc: An HIV EntryInhibitor Targeting CCR5. Retrovirology 2(Suppl. 1): S13). In someaspects, the CCR5 antagonist is a spiropiperidine. CCR5 antagonist.(Rotstein D. M. et al. 2009. Spiropiperidine CCR5 antagonists.Bioorganic & Medicinal Chemistry Letters. 19 (18): 5401-5406. In someembodiments, the CCR5 antagonist is INCB009471 (Kuritzkes, D. R. 2009.HIV-1 entry inhibitors: an overview. Curr. Opin. HIV AIDS. 4(2): 82-7).

In a preferred embodiment the dual MET and VEGFR2 inhibitor is selectedfrom the group consisting of Cabozantinib, Foretinib and E7050.

In another aspect, the treatment is an mTor inhibitor. In some aspects,the mTor inhibitor is a dual mTor/PI3kinase inhibitor. In some aspects,the mTor inhibitor is used to prevent or inhibit metastasis. In someaspects the mTor inhibitor is selected, from the group consisting of:ABI009 (sirolimus), rapamycin (sirolimus), Abraxane (paclitaxel), Absorb(everolimus); Afinitor (everolimus), Afinitor with Gleevec, AS703026(pimasertib), Axxess (umirolimus), AZD2014, BEZ235, Biofreedom(umirolimus), BioMatrix (umirolimus), BioMatrix flex (umirolimus),CC115, CC223, Combo Bio-engineered Sirolimus Eluting Stent ORBUSNEICH(sirolimus), Curaxin CBLC102 (mepacrine), DE109 (sirolimus), DS3078,Endeavor DES (zotarolimus), Endeavor Resolute (zotarolimus); Femara(letrozole), Hocena (antroquinonol), INK128, Inspiron (sirolimus),IPI504 (retaspimycin hydrochloride), KRN951 (tivozanib), ME344, MGA031(teplizumab), MiStent SES (sirolimus), MKC1, Nobori (umirolimus),OSI027, OVI123 (cordycepin), Palomid 529, PF04691502, Promus Element(everolimus), PWT33597, Rapamune (sirolimus), Resolute DES(zotarolimus), RG7422, SAR245409, SF1126, SGN75 (vorsetuzumabmafodotin), Synergy (everolimus), Taltorvic (ridaforolimus), Tarceva(erlotinib), Torisel (temsirolimus), Xience Prime (everolimus), Xience V(everolimus), Zomaxx (zotarolimus), Zortress (everolimus), ZotarolimusEluting Peripheral Stent MEDTRONIC (zotarolimus), AP23841, AP24170,ARmTOR26, BN107, BN108, Canstatin GENZYME (canstatin), CU906, EC0371,EC0565, KI1004, LOR220, NV128, Rapamycin ONCOIMMUNE (sirolimus), SB2602,Sirolinius PNP SAMYANG BIOPHARMACEUTICALS (sirolimus), TOP216, VLI27,VS5584, WYE125132, XL388, Advacan (everolimus); AZD8055, Cypher SelectPlus Sirolimus eluting Coronary Stent (sirolimus), Cypher Sirolimuseluting coronary stent (sirolimus), Drug Coated Balloon (sirolimus),E-Magic Plus (sirolimus), Emtor (sirolimus), Esprit (everolimus),Evertor (everolimus), HBF0079, LCP-Siro (sirolimus), Limus CLARIS(sirolimus), mTOR Inhibitor CELLZOME, Nevo Sirolimus eluting CoronaryStent (sirolimus), nPT-mTOR, Rapacan (sirolimus), Renacept (sirolimus),ReZolve (sirolimus), Rocas (sirolimus), SF1126, Sirolim (sirolimus),Sirolimus NORTH CHINA (sirolimus), Sirolimus RANBAXY (sirolimus),Sirolimus WATSON (sirolimus), Siropan (sirolimus), Sirova (sirolimus),Supralimus (sirolimus), Supralimus-Core (sirolimus), Tacrolimus WATSON(tacrolimus), TAFA93, Temsirolimus ACCORD (temsirolimus), TemsirolimusSANDOZ (temsirolimus), TOP216, Xience Prime, (everolimus), Xience V(everolimus). In a specific aspect the mTor inhibitor is Afinitor(everolimus)(http://www.afinitor.com/index.jsp?usertrack.filter_applied=true&NovaId=4029462064338207963; last accessed Nov. 28, 2012). In another aspect, mTorinhibitors can be identified through methods known in the art. (See,e.g., Zhou, H. et al. Updates of mTor inhibitors. 2010. AnticancerAgents Med. Chem. 10(7): 571-81, which is herein incorporated byreference). In some aspects, the mTor inhibitor is used to treat orprevent or inhibit metastasis in a patient with advanced lung cancer. Insome aspects, the mTor inhibitor is used in combination with a secondtreatment. In some aspects, the second treatment is any treatmentdescribed herein.

In another aspect, the treatment is a Src kinase inhibitor. In someaspects, the Src inhibitor is used to prevent or inhibit metastasis. Insome aspects, the Src kinase inhibitor is selected from the group:AZD0530 (saracatinib), Bosulif (bosutinib), ENMD981693, KD020, KX01,Sprycel (dasatinib), Yervoy (ipilimumab), AP23464, AP23485, AP23588,AZD0424, c-Src Kinase Inhibitor KISSEI, CU201, KX2361, SKS927, SRN004,SUNK706, TG100435, TG100948, AP23451, Dasatinib HETERO (dasatinib),Dasatinib VALEANT (dasatinib), Fontrax (dasatinib), Src Kinase InhibitorKINEX, VX680, (tozasertib lactate), XL228, and SUNK706. In someembodiments, the Src kinase inhibitor is dasatinib. In another aspect,Src kinase inhibitors can be identified through methods known in the art(See, e.g., Sen, B. and Johnson, F. M. Regulation of Src Family Kinasesin Human Cancers. 2011, J. Signal Transduction. 2011: 14 pages, which isherein incorporated by reference). In some aspects, the Src kinaseinhibitor is used to treat or prevent or inhibit metastasis in a patientthat is positive for the SRC-responsive signature (SRS). In someaspects, the patient is SRS+(See, e.g., Zhang, C H. F, et al. LatentBone Metastasis in Breast Cancer Tied to Src-Dependent survival signals.2009. Cancer Cell. 16:67-78, which is herein incorporated by reference.)In some aspects, the Src kinase inhibitor is used to treat or prevent orinhibit metastasis in a patient with advanced lung cancer. In someaspects, the Src kinase inhibitor is used in combination with a secondtreatment. In some aspects, the second treatment is any treatmentdescribed herein.

In another aspect, the treatment is a COX-2 inhibitor. In some aspects,the COX-2 inhibitor is used to prevent or inhibit metastasis. In someaspects, the COX-2 inhibitor is selected from the group: ABT963,Acetaminophen ER JOHNSON (acetaminophen), Acular X (ketorolactromethamine), BAY1019036 (aspirin), BAY987111 (diphenhydramine,naproxen sodium), BAY11902 (piroxicam), BCIBUCH001 (ibuprofen);Capoxigem (apricoxib), CS502, CS670 (pelubiprofen), Diclofenac HPBCD(diclofenac), Diractin (ketoprofen), GW406381, HCT1026(nitroflurbiprofen), Hyanalgese-D (diclofenac), HydrocoDex(acetaminophen, dextromethorphan, hydrocodone), Ibuprofen Sodium PFIZER(ibuprofen sodium), Ibuprofen with Acetaminophen PFIZER (acetaminophen,ibuprofen), Impracor (ketoprofen), IP880 (diclofenac), IP940(indomethacin), ISV205 (diclofenac, sodium), JNS013 (acetaminophen,tramadol hydrochloride), Ketoprofen TDS (ketoprofen), LTNS001 (naproxenetemesil), Mesalamine SAL1X (mesalamine), Mesalamine SOFAR (mesalamine),Mesalazine (mesalamine), ML3000 (licofelone), MRX7EAT (etodolac),Naproxen IROKO (naproxen), NCX4016 (nitroaspirin), NCX701(nitroacetaminophen), Nuprin SCOLR (ibuprofen), OMS103HP (amitriptylinehydrochloride, ketoprofen, oxymetazoline hydrochloride), Oralease(diclofenac), OxycoDex (dextromethorphan, oxycodone), P54, PercoDex(acetaminophen, dextromethorphan, oxycodone), PL3100 (naproxen,phosphatidyl choline), PSD508, R-Ketoprofen (ketoprofen), Remura(bromfenac sodium), ROX828 (ketorolac tromethamine), RP19583 (ketoprofenlysine), RQ00317076, SDX101 (R-etodolac), TDS943 (diclofenac sodium),TDT070 (ketoprofen), TPR100, TQ1011 (ketoprofen), TT063(S-flurbiprofen), UR8880 (cimicoxib), V0498TA01A (ibuprofen), VT122(etodolac, propranolol), XP20B (acetaminophen, dextropropoxyphene),XP21B (diclofenac potassium), XP21L (diclofenac potassium), Zoenasa(acetylcysteine, mesalamine), Acephen, Actifed Plus, Actifed-P, Acular,Acular LS, Acular PF, Acular X, Acuvail, Advil, Advil Allergy Sinus,Advil Cold and Sinus, Advil Congestion Relief, Advil PM, Advil PMCapsule, Air Salonpas, Airtal, Alcohol-Free NyQuil Cold & Flu Relief,Aleve, Aleve ABDI IBRAHIM, Aleve-D, Alka-Seltzer, Alka-Seltzer BAYER,Alka-Seltzer Extra Strength, Alka-Seltzer Lemon-Lime, Alka-SeltzerOriginal, Alka-Seltzer Plus, Alka-Seltzer plus Cold and Cough,Alka-Seltzer plus Cold and Cough Formula, Alka-Seltzer Plus Day andNight Cold Formula, Alka-Seltzer Plus Day Non-Drowsy Cold Formula,Alka-Seltzer Plus Flu Formula, Alka-Seltzer Plus Night Cold Formula,Alka-Seltzer Plus Sinus Formula, Alka-Seltzer Plus Sparkling OriginalCold Formula, Alka-Seltzer PM, Alka-Seltzer Wake-Up Call, Anacin,Anaprox, Anaprox MINERVA, Ansaid, Apitoxin, Apranax, Apranax abdi,Arcoxia, Arthritis Formula Bengay, Arthrotec, Asacol, Asacol HD, AsacolMEDUNA ARZNEIMITTEL, Asacol ORIFARM, Aspirin BAYER, Aspirin Complex,Aspirin Migran, AZD3582, Azulfidine, Baralgan M, BAY1019036, BAY987111,BAY11902, BCIBUCH001, Benadryl Allergy, Benadryl Day and Night, Benylin4 Flu, Benylin Cold and Flu, Benylin Cold and Flu Day and Night, BenylinCold and Sinus Day and Night, Benylin Cold and Sinus Plus, Benylin Danand Night Cold and Flu Relief, Benylin1 All-In-One, Brexin, BrexinANGELINI, Bromday, Bufferin, Buscopan Plus, Caldolor, Calmatel, Cambia,Canasa, Capoxigem, Cataflam, Celebrex, Celebrex ORIFARM, Children'sAdvil Allergy Sinus, Children's Tylenol, Children's Tylenol Cough andRunny Nose, Children's Tylenol plus cold, Children's Tylenol-plus Coldand Cough, Children's Tylenol plus cold and stuffy nose, Children'sTylenol plus Flu, Children's Tylenol plus cold & allergy, Children'sTylenol plus Cough & Runny Nose, Children's Tylenol plus Cough & SoreThroat, Children's Tylenol, plus multi symptom cold, Clinoril, CodralCold and Flu, Codral Day and Night Day Tablets, Codral Day and NightNight Tablets, Codral Nightime, Colazal, Combunox, Contac Cold plus Flu,Contac Cold plus Flu Non-Drowsy, Coricidin D, Coricidin HBP Cold andFlu, Coricidin HBP Day and Night Multi-Symptom Cold, Coricidin HBPMaximum Strength Flu, Coricidin HBP Nighttime Multi-Symptom Cold,Coricidin II Extra Strength Cold and Flu, CS502, CS670, Daypro, DayproAlta, DDS06C, Demazin Cold and Flu, Demazin Cough, Cold and Flu, Demazinday/night Cold and Flu, Demazin PE Cold and Flu, Demazin PE day/nightCold and Flu, Diclofenac HPBCD, Dimetapp Day Relief, DimetappMulti-Symptom Cold and Flu, Dimetapp Night Relief, Dimetapp Pain andFever Relief, Dimetapp PE Sinus Pain, Dimetapp PE Sinus Pain plusAllergy, Dipentum, Diractin, Disprin Cold 'n' Fever, Disprin Extra,Disprin Forte, Disprin Plus, Dristan Cold, Dristan Junior, DrixoralPlus, Duexis, Dynastat, Efferalgan, Efferalgan Plus Vitamin C,Efferalgan Vitamin C, Elixsure IB, Excedrin Back and Body, ExcedrinMigraine, Excedrin PM, Excedrin Sinus Headache, Excedrin TensionHeadache, Falcol, Fansamac, Feldene, FeverAll, Fiorinal, Fiorinal withCodeine, Flanax, Flector Patch, Flucam, Fortagesic, Gerbin, Giazo,Gladio, Goody's Back and Body Pain; Goody's Cool Orange, Goody's ExtraStrength; Goody's PM, Greaseless Bengay, GW406381, HCT1026, He Xing Yi,Hyanalgese-D, HydrocoDex, Ibuprofen Sodium PFIZER, Ibuprofen with,Acetaminophen PFIZER, Icy Hot SANOFI AVENTIS, Impracor, Indocin,Indomethacin APP PHARMA, Indomethacin MYLAN, Infants' Tylenol, IP880,IP940, Iremod, ISV205, JNS013, Jr. Tylenol, Junifen, Junior StrengthAdvil, Junior Strength Motrin, Ketoprofen TDS, Lemsip Max, Lemsip MaxAll in One, Lemsip Max All Night, Lemsip Max Cold and Flu, Lialda,Listerine Mouth Wash, Lloyds Cream, Lodine, Lorfit P, Loxonin, LTNS001,Mersyndol, Mesalamine SALIX, Mesalamine SOFAR, Mesalazine, MesasalGLAXO, Mesasal SANOFI, Mesulid, Metsal Heat Rub, Midol Complete, MidolExtended Relief, Midol Liquid Gels, Midol PM, Midol Teen Formula,Migranin COATED TABLETS, ML3000, Mobic, Mohrus, Motrin, Motrin Cold andSinus Pain, Motrin PM, Movalis ASPEN, MRX7EAT, Nalfon, Nalfon PEDINOL,Naprelan, Naprosyn, Naprosyn RPG LIFE SCIENCE, Naproxen IROKO, NCX4016,NCX701, NeoProfen LUNDBECK, Nevanac, Nexcede, Niflan, Norgesic MEDICIS,Novalgin, Nuprin SCOLR, Nurofen, Nurofen Cold and Flu, Nurofen MaxStrength Migraine, Nurofen Plus, Nuromol, NyQuil with Vitamin C, Ocufen,OMS103HP, Oralease, Orudis ABBOTT JAPAN, Oruvail, Osteluc, OxycoDex,P54, Panadol, Panadol Actifast, Paradine, Paramax, Parfenac, Pedea,Pennsaid, Pentasa, Pentasa ORIFARM, Peon, Percodan, Percodan-Demi,PercoDex, Percogesic, Perfalgan, PL2200, PL3100, Ponstel, Prexige,Prolensa, PSD508, R-Ketoprofen, Rantudil, Relafen, Remura, Robaxisal,Rotec, Rowasa, ROX828, RPI9583, RQ00317076, Rubor, Salofalk, Salonpas,Saridon, SDX101, Seltouch, sfRowasa, Shinbaro, Sinumax, Sinutab,Sinutab, sinus, Spalt, Sprix, Strefen, Sudafed Cold and Cough, SudafedHead Cold and Sinus, Sudafed PE Cold plus Cough, Sudafed PE Pressureplus Pain, Sudafed PE, Severe Cold, Sudafed PE Sinus Day plus NightRelief Day Tablets, Sudafed PE Sinus Day plus Night Relief NightTablets, Sudafed PE Sinus plus Anti-inflammatory Pain Relief, SudafedSinus Advance, Surgam, Synalgos-DC, Synflex, Tavistallergy/sinus/headache, TDS943, TDT070, Theraflu Cold and Sore Throat,Theraflu Daytime Severe Cold and Cough, Theraflu Daytime Warming Relief,Theraflu Warming, Relief Caplets Daytime Multi-Symptom Cold, TherafluWarming Relief Cold and Chest Congestion, Thomapyrin, Thomapyrin C,Thomapyrin Effervescent, Thomapyrin Medium, Tilcotil, Tispol, Tolectin,Toradol, TPR100, TQ1011, Trauma-Salbe, Trauma-Salbe Kwizda, Treo,Treximet, Trovex, TT063, Tylenol, Tylenol Allergy Multi-Symptom, TylenolBack Pain, Tylenol Cold & Cough Daytime, Tylenol Cold & Cough Nighttime,Tylenol Cold and Sinus Daytime, Tylenol Cold and Sinus Nighttime,Tylenol Cold Head Congestion Severe, Tylenol Cold Multi Symptom Daytime,Tylenol Cold Multi Symptom Nighttime Liquid, Tylenol Cold Multi SymptomSevere, Tylenol Cold Non-Drowsiness Formula, Tylenol Cold SevereCongestion Daytime, Tylenol Complete Cold, Cough and Flu Night time,Tylenol Flu Nighttime, Tylenol Menstrual, Tylenol PM, Tylenol SinusCongestion & Pain Daytime, Tylenol Sinus Congestion & Pain Nighttime,Tylenol Sinus Congestion & Pain Severe, Tylenol Sinus Severe CongestionDaytime, Tylenol Ultra Relief, Tylenol with Caffeine and Codeinephosphate, Tylenol with Codeine phosphate, Ultra Strength Bengay Cream,Ultracet, UR8880, V0498TA01A, Vicks NyQuil Cold and Flu Relief,Vicoprofen Vimovo, Voltaren Emulgel, Voltaren GEL, Voltaren NOVARTISCONSUMER HEALTH GMBH, Voltaren XR, VT122, Xefo, Xefo Rapid, Xefocam,Xibrom, XL3, Xodol, XP20B, XP21B, XP21L, Zipsor, and Zoenasa. In anotheraspect, COX-2 inhibitors can be identified through methods known in theart (See, e.g., Dannhardt, G. and Kiefer, W. Cyclooxygenaseinhibitors—current status and future prospects, 2001. Eur. J. Med. Chem,36:109-126, which is herein incorporated by reference). In some aspects,the COX-2 inhibitor is used to treat or prevent, or inhibit metastasisin a patient with advanced lung cancer. In some aspects, the COX-2inhibitor is used in combination with a second treatment. In someaspects, the second treatment is any treatment described herein. In someaspects, the COX-2 inhibitor is used in combination with a secondtreatment selected from the group consisting of: Denosumab, Zometa(http://www.us.zometa.com/index.jsp?usertrack.filter_applied=true&NovaId=2935376934467633633; last accessed Dec. 2, 2012), Carbozantinib or Cabozantinib,Antibody or peptide blocking PTHLH (parathyroid hormone like hormone) orPTHrP (parathyroid hormone related protein).

In a preferred embodiment the Radium 223 therapy is Alpharadin (aka,Xofigo) (radium-223 dichloride). Alpharadin uses alpha radiation fromradium-223 decay to kill cancer cells. Radium-223 naturally self-targetsto bone metastases by virtue of its properties as a calcium-mimic. Alpharadiation has a very short range of 2-10 cells (when compared to currentradiation therapy which is based on beta or gamma radiation), andtherefore causes less damage to surrounding healthy tissues(particularly bone marrow). With similar properties to calcium,radium-223 is drawn to places where calcium is used to build bone in thebody, including the site of faster, abnormal bone growth—such as thatseen in the skeletal metastases of men with advanced,castration-resistant prostate cancer. Radium-223, after injection, iscarried in the bloodstream to sites of abnormal bone, growth. The placewhere a cancer starts in the body is known as the primary tumor. Some ofthese cells may break away and be carried in the bloodstream to anotherpart of the body. The cancer cells may then settle in that part of thebody and form a new tumor. If this happens it is called a secondarycancer of a metastasis. Most patients with late stage prostate cancersuffer the maximum burden of disease in their bones. The aim withradium-223 is to selectively target this secondary cancer. Anyradium-223 not taken-up in the bones is quickly routed to the gut andexcreted. In a preferred embodiment, the agent preventing the bonedegradation is a bisphosphonate. In a yet more preferred embodiment, thebisphosphonate is the zoledronic acid.

Alternatively a combined treatment can be carried out in which more thanone agent from those mentioned above are combined to treat and/orprevent the metastasis or said agents can be combined with othersupplements, such as calcium or vitamin D or with a hormone treatment.

Method of Diagnosis or Prognosis of Metastasis, in Lung Cancer Based onDetecting the Amplification of the c-MAF Gene

In one aspect, the invention relates to an in-vitro method for thediagnosis of metastasis in a subject with lung cancer (hereinafter,fourth diagnosis method of the invention) and/or for the prognosis ofthe tendency to develop metastasis in a subject with lung cancer whichcomprises determining if the c-MAF gene is amplified in a tumor tissuesample of said subject; wherein if said gene is amplified with respectto a control sample, then said subject has a positive diagnosis formetastasis or a greater tendency to develop metastasis.

In a particular embodiment, the lung cancer diagnosed in the fourthmethod of the invention is NSCLC.

The terms “c-MAF gene”, “metastasis”, “tumor sample”, “lung cancer”,“diagnosis of metastasis in a subject with lung cancer”, “prognosis ofthe tendency to develop metastasis in a subject with lung cancer”,“subject”, “patient”, “subject having a positive diagnosis ofmetastasis”, “subject having a greater tendency to develop metastasis”have been described in detail in the context of the first method of theinvention and are equally applicable to the fourth method of theinvention.

In a particular embodiment, the degree of amplification of the c-MAFgene can be determined by means of determining the amplification of achromosome region containing said gene. Preferably, the chromosomeregion the amplification of which is indicative of the existence ofamplification of the c-MAF gene is the locus 16q22-q24 which includesthe c-MAF gene. The locus 16q22-q24 is located in chromosome 16, in thelong arm of said chromosome and in a range between band 22 and band 24.This region corresponds in the NCBI database with the contigsNT_010498.15 and NT_010542.15. In another preferred embodiment, thedegree of amplification of the c-MAF gene can be determined by means ofusing a probe specific for said gene.

The fourth diagnosis/prognosis method of the invention comprises, in afirst step, determining if the c-MAF gene is-amplified in a tumor sampleof a subject. To that end, the amplification of the c-MAF gene in thetumor sample is compared with respect to a control sample.

The term, “amplification of a gene” as understood herein refers to aprocess through which various copies of a gene or of a gene fragment areformed in an individual cell or a cell line. The copies of the gene arenot necessarily located in the same chromosome. The duplicated region isoften called an “amplicon”. Normally, the amount of mRNA produced, i.e.,the gene expression level also increases in proportion to the copynumber of a particular gene.

In a particular embodiment, the fourth method of the invention for thediagnoses of metastasis in a subject with lung cancer and/or for theprognosis of the tendency to develop metastasis in a subject with lungcancer, comprises determining the c-MAF gene copy number in a tumorsample of said subject and comparing said copy number with the copynumber of a control or reference sample, wherein if the c-MAF copynumber is greater with respect to the c-MAF copy number of a controlsample, then the subject has a positive diagnosis of metastasis or agreater tendency to develop metastasis.

The control sample refers to a tumor sample of a subject with lungcancer who has not suffered metastasis or that correspond to the medianvalue of the c-MAF gene copy number measured in a tumor tissuecollection in biopsy samples of subjects with lung cancer who have notsuffered metastasis. Said reference sample is typically obtained bycombining equal amounts of samples from a subject population. If thec-MAF gene copy number is increased with respect to the copy number ofsaid gene in the control sample then subject has a positive diagnosisfor metastasis or a greater tendency to develop metastasis.

As used herein, the term “gene copy number” refers to the copy number ofa nucleic acid molecule in a cell. The gene copy number includes thegene copy number in the genomic (chromosomal) DNA of a cell. In a normalcell (non-tumoral cell), the gene copy number is normally two copies(one copy in each member of the chromosome pair). The gene copy numbersometimes includes half of the gene copy number taken from samples of acell population.

In the present invention, “increased gene copy number” is understood aswhen the c-MAF gene copy number is more than the copy number that areference sample or control sample has. In particular, it can beconsidered that a sample has an increased c-MAF copy number when thecopy number is more than 2 copies, for example, 3, 4, 5, 6, 7, 8, 9 or10 copies, and even more than 10 copies of the c-MAF gene.

In some embodiments, the amplification is in region at the 16q23 locus.In some embodiments, the amplification is in any part of the chromosomalregion between about Chr. 16—about 79,392,959 bp to about 79,663,806 bp(from centromere to telomere). In some embodiments, the amplification isin the genomic region between about Chr. 16—about 79,392,959 bp to about79,663,806 bp, but excluding DNA repeating elements. In someembodiments, amplification is measured using a probe specific for thatregion.

In a particular embodiment, the amplification or the copy number isdetermined by means of in situ hybridization or PCR.

Methods for determining whether the c-MAF gene or the chromosome region16q22-q24 is amplified are widely known in the state of the art. Saidmethods include, without limitation, in situ hybridization (ISH) (suchas fluorescence in situ hybridization (FISH), chromogenic in situhybridization (CISH) or silver in situ hybridization (SISH)), genomiccomparative hybridization or polymerase-chain reaction (such as realtime quantitative PCR). For any ISH method, the amplification or thecopy number can be determined by counting the number of fluorescentpoints, colored points or points with silver in the chromosomes or inthe nucleus.

The fluorescence in situ hybridization (FISH) is a cytogenetic techniquewhich is used for detecting and locating the presence or absence ofspecific DNA sequences in chromosomes. FISH uses fluorescence probeswhich only bind to some parts of the chromosome with which they show ahigh degree of sequence similarity. In a typical FISH method, the DNAprobe is labeled with a fluorescent molecule or a hapten, typically inthe form of fluor-dUTP; digoxigenin-dUTP, biotin or hapten-dUTP which isincorporated in the DNA using enzymatic reactions, such as nicktranslation or PCR, The sample containing the genetic material (thechromosomes) is placed on glass slides and is denatured by a form amidetreatment. The labeled probe is then hybridized with the samplecontaining the genetic material under suitable conditions which will bedetermined by the person skilled in the art. After the hybridization,the sample is viewed either directly (in the case of a probe labeledwith fluorine) or indirectly (using fluorescently labeled antibodies todetect the hapten),.

In the case of CISH, the probe is labeled with digoxigenin, biotin orfluorescein and is hybridized with the sample containing the geneticmaterial in suitable conditions.

Any marking or labeling molecule which can bind to a DNA can be used tolabel the probes used in the fourth method of the invention, thusallowing the detection of nucleic acid molecules. Examples of labels forthe labeling include, although not limited to, radioactive isotopes,enzyme, substrates, cofactors, ligands, chemiluminescence agents,fluorophores, haptens, enzymes and combinations thereof. Methods forlabeling and guideline for selecting suitable labels for differentpurposes can be found, for example, in Sambrook et al. (MolecularCloning: A Laboratory Manual, Cold Spring Harbor, N.Y., 1989) andAusubel et al. (In Current Protocols in Molecular Biology, John Wileyand Sons, New York, 1998).

Once the existence of amplification is determined, either by directlydetermining the amplification of the c-MAF gene or by determining theamplification of the locus 16q22-q24, and after being compared with theamplification of said gene in the control sample, if amplification inthe c-MAF gene is detected, it is indicative of the fact that thesubject has a positive diagnosis for metastasis or a greater tendency todevelop metastasis.

The determination of the amplification of the c-MAF gene needs to becorrelated with values of a control sample or reference sample thatcorrespond to the level of amplification of the c-MAF gene measured in atumor tissue sample of a subject with lung cancer who has not sufferedmetastasis or that correspond to the median value of the amplificationof the c-MAF gene measured in a tumor tissue collection in biopsysamples of subjects with lung cancer who have not suffered metastasis.Said reference sample is typically obtained by combining equal amountsof samples from a subject population. In general, the typical referencesamples will be obtained from subjects who are clinically welldocumented and in whom the absence of metastasis is well characterized.The sample collection from which the reference level is derived willpreferably be made up of subjects suffering the same type of cancer asthe patient object of the study (e.g., NSCLC). Once this median valuehas been established, the level of amplification of c-MAF in tumortissues of patients can be compared with this median value, and thus, ifthere is amplification, the subject has a positive diagnosis ofmetastasis or a greater tendency to develop metastasis.

In a preferred embodiment, the metastasis is bone metastasis. In a yetmore preferred embodiment, the bone metastasis is osteolytic bonemetastasis. As used herein, the expression “osteolytic bone metastasis”refers to a type of metastasis in which bone resorption (progressiveloss of bone density) is produced in the proximity of the metastasisresulting from the stimulation of the osteoclast activity by the tumorcells and is characterized by severe pain, pathological fractures,hypercalcaemia, spinal cord compression and other syndromes resultingfrom nerve compression.

Method of Prognosis of Metastasis in Lung Cancer Based on Detecting theTranslocation of the c-MAF Gene

In another aspect, the invention relates to an in vitro method forpredicting the clinical outcome of a patient suffering from lung cancer,which comprises determining if the c-MAF gene is translocated in asample of said subject wherein a translocation of the c-MAF gene isindicative of a poor clinical outcome.

In another aspect, the invention relates to an in vitro method forpredicting the clinical outcome of a patient suffering lung cancer,which comprises determining if the c-MAF gene is translocated in asample of said subject wherein a translocation of the c-MAF gene isindicative of a poor clinical outcome.

In some embodiments, the translocated gene is from the region at the16q23 locus. In some embodiments, the translocated gene is from any partof the chromosomal region between about Chr. 16—about 79,392,959 bp toabout 79,663,806 bp (from centromere to telomere). In some embodiments,the translocated gene is from the genomic region between about Chr.16—about 79,392,959 bp to about 79,663,806 bp, but excluding DNArepeating elements. In some embodiments, the translocation is measuredusing a probe specific for that region.

In a particular embodiment, the translocation of the c-MAF gene can bedetermined by means of determining the translocation of a chromosomeregion containing said gene. In one embodiment, the translocation is thet(14,16) translocation. In another embodiment, the chromosome regionthat is translocated is from locus 16q22-q24. The locus 16q22-q24 islocated in chromosome 16, in the long arm of said chromosome and in arange between band 22 and band 24. This region corresponds in the NCBIdatabase with the contigs NT_010498.15 and NT_010542.15. In a preferredembodiment, the c-MAF gene translocates to chromosome 14 at the locus14q32, resulting in the translocation t(14,16)(q32,q23). Thistranslocation places the MAF gene next to the strong enhancers in theIgH locus, which, in some cases, leads to overexpression of MAF.(Eyehene, A., Rocques, N., and Puoponnot, C., A new MAFia in cancer.2008. Nature-Reviews: Cancer. 8:683-693.)

In a preferred embodiment, the translocation of the c-MAF gene can bedetermined by means of using a probe specific for said translocation. Insome embodiments, the translocation is measured using a dual colorprobe. In some embodiments, the translocation is measured using a dualfusion probe. In some embodiments, the translocation is measured using adual color, dual fusion probe. In some embodiments, the translocation ismeasured using two separate probes.

In another preferred embodiment, the translocation of the C-MAF gene isdetermined using the Vysis LSI IGH/MAF Dual Color dual fusion probe(http://www.abbottmolecular.com/us/products/analyte-specific-reagent/fish/vysis-Isi-igh-maf-dual-color-dual-fusion-probe.html; last accessed Nov. 5, 2012), whichcomprises a probe against 14q32 and 16q23. In another preferredembodiment, the translocation of the c-MAF gene is determined using aKreatech diagnostics MAF/IGH gt(14;16) Fusion probe(http://www.kreatech.com/products/repeat-freetm-poseidontm-fish-probes/hematology/maf-igh-gt1416-fusion-probe.html; last accessed Nov. 5, 2012), an AbnovaMAF FISH probe(http://www.abnova.com/products/products_detail.asp?Catalog_id=FA0375;last accessed Nov. 5, 2012), a Cancer Genetics Italia IGH/MAF Two Color,Two Fusion translocation probe(http://www.cancergeneticsitalia.com/dna-fish-probe/ighmaf/; lastaccessed Nov. 5, 2012), a Creative Bioarray IGH/MAF-t(14;16) (q32;q23)FISH probe(http://www.creative-bioarray.com/products.asp?cid=35&page=10; lastaccessed Nov. 5, 2012), a Arup Laboratories multiple myeloma panel byFISH(http://www.aruplab.com/files/technical-bulletins/multiple%20Myeloma%20%28MM%29%20by%20FISH.pdf;last accessed Nov. 5, 2012), an Agilent probe specific to 16q23 or 14q32(http://www.genomics.agilent.com/ProductSearch.aspx?chr=16&start=79483700&end=79754340; last accessed Nov. 5, 2012;http://www.genomics.agilent.com/ProductSearch.aspx?Pageid=3000&ProductID=637;last accessed Nov. 5, 2012), a Dako probe specific to 16q23 or 14q32(http://www.dako.com/us/ar42/psg42806000/baseproducts_surefish.htm?setCountry=true&purl=ar42/psg42806000/baseproducts_surefish.htm?undefined&submit=Accept%20country; last accessed Nov. 5, 2012), a Cytocell IGH/MAF Translocation,Dual Fusion Probe(http://www.zentech.be/uploads/docs/products_info/prenatalogy/cytocell%202012-2013%20catalogue%5B3%5D.pdf;last accessed Nov. 5, 2012), a Metasystems XL IGH/MAF Translocation DualFusion Probe(http://www.metasystems-international.com/index.php?option=com_joodb&view=article&joobase=5&id=12%3Ad-5029-100-og&Itemid=272;last accessed Nov. 5, 2012), a Zeiss FISH Probes XL, 100 μl, IGH/MAFB(https://www.micro-shop.zeiss.com/?s=440675675dedc6&1=en&p=uk&f=r&i=5000&o=&h=25&n=1U2d=000000-0528-231-uk; last accessed Nov. 5, 2012) or a Genycell BiotechIGH/MAF Dual Fusion Probe(http://www.google.com/url?sa=t&ret=j&q=&esrc=s&source=web&cd=1&ved=0CCQQFjAA&url=http%3A%2F%2Fwww.genycell.es%2Fimages%2Fproductos%2Fbrochures%2Flphmie6_86.ppt&ci=MhGYUOi3GKWH0QGlt4DoDw&usg=AFQjCNEqQMbT8vQGjJbi9riEf31VgoFTFQ&sig2=V5IS8juEMVHB18Mv2Xx_Ww; last accessed Nov. 5,2012)

In some embodiments, the label on the probe is a fluorophore. In someembodiments, the fluorophore on the probe is orange. In someembodiments, the fluorophore on the probe is green. In some embodiments,the fluorophore on the probe is red. In some cases, the fluorophore onthe probe is yellow. In some embodiments, one probe is labeled with ared fluorophore, and one with a green fluorophore. In some embodiments,one probe is labeled with a green fluorophore and one with an orangefluorophore. In some cases, the fluorophore on the probe is yellow. Forinstance, if the MAF-specific probe is labeled with a red fluorophore,and the IGH-specific probe is labeled with a green fluorophore, if whiteis seen it indicates that the signals overlap and translocation hasoccurred.

In some embodiments, the fluorophore is SpectrumOrange. In someembodiments, the fluorophore is SpectrumGreen. In some embodiments, thefluorophore is DAPI. In some embodiments, the fluorophore isPlatinumBright405. In some embodiments, the fluorophore isPlatinumBright415. In some embodiments, the fluorophore isPlatinumBright495. In some embodiments, the fluorophore isPlatinumBright505. In some embodiments, the fluorophore isPlatinumBright550. In some embodiments, the fluorophore isPlatinumBright547. In some embodiments, the fluorophore isPlatinumBright570. In some embodiments, the fluorophore isPlatinumBright590. In some embodiments, the fluorophore isPlatinumBright647. In some embodiments, the fluorophore isPlatinumBright495/550. In some embodiments, the fluorophore isPlatinumBright415/495/550. In some embodiments, the fluorophore isPAPI/PlatinumBright495/550. In some embodiments, the fluorophore isFITC. In some embodiments, the fluorophore is Texas Red. In someembodiments, the fluorophore is DEAC. In some embodiments, thefluorophore is R6G. In some embodiments, the fluorophore is Cy5. In someembodiments, the fluorophore is FITC, Texas Red and DAPI. In someembodiments, a DAPI counterstain is used to visualize the translocation,amplification or copy number alteration.

One embodiment of the invention comprises a method in which in a firststep it is determined if the c-MAF gene is translocated in a sample of asubject. In a preferred embodiment, the sample is a tumor tissue sample.

In a particular embodiment, a method of the invention for the prognosisof the tendency to develop bone metastasis in a subject with lung cancercomprises determining the c-MAF gene copy number in a sample of saidsubject wherein the c-MAF gene is translocated and comparing said copynumber with the copy number of a control or reference sample, wherein ifthe c-MAF copy number is greater with respect to the c-MAF copy numberof a control sample, then the subject has a greater tendency to developbone metastasis. In a preferred embodiment, the bone metastasis is veryearly bone metastasis. In a preferred embodiment, the bone metastasis isosteolytic metastasis.

“Average level” as used herein relates to a single value of c-MAFexpression level (as a mean, mode, or median) that summarizes orrepresents the general significance of a set of unequal values. In apreferred embodiment the average level corresponds to the average ofexpression levels obtained from a representative cohort of lung cancertumors. The patient cohort is defined by age that is representative ofthe individual patient that one is attempting to evaluate.

“Standard deviation” as used herein relates to a measure of thedispersion of a collection of numbers. For example, the standarddeviation for the average normal level of c-MAF is the dispersion of acollection of the c-MAF levels found in lung tumor samples The morespread apart the data, the higher the deviation. Standard deviation canbe obtained by extracting the square root of the mean of squareddeviations of observed values from their mean in a frequencydistribution.

Once the c-MAF gene expression level in a sample from a subject withlung cancer, has been measured and compared with the average level, ifthe expression level of said gene is above the average plus one standarddeviation with respect to the average level, then it can be concludedthat said subject has a greater tendency to develop early bonemetastasis.

Methods for determining whether the c-MAF gene or the chromosome region16q22-q24 is translocated are widely known in the state of the art andinclude those described previously for the amplification of c-MAF. Saidmethods include, without limitation, in situ hybridization (ISH) (suchas fluorescence in situ hybridization (FISH), chromogenic in situhybridization (CISH) or silver in situ hybridization (SISH)), genomiccomparative hybridization or polymerase chain reaction (such as realtime quantitative PCR). For any ISH method, the amplification, the copynumber, or the translocation can be determined by counting the number offluorescent points, colored points or points with silver in thechromosomes or in the nucleus. In other embodiments, the detection ofcopy number alterations and translocations can be detected through theuse of whole genome sequencing, exome sequencing or by the use of anyPCR derived technology. For instance, PCR can be performed oh samples ofgenomic DNA to detect translocation. In one embodiment, quantitative PCRis used. In one embodiment, PCR is performed with a primer specific tothe c-MAF gene and a primer specific to the IGH promoter region; if aproduct is produced, translocation has occurred.

In some embodiments, the amplification and copy number of the c-MAF geneare determined after translocation of the c-MAF gene is determined. Insome embodiments, the probe is used to determine if the cell ispolyploid for the c-MAF gene. In some embodiments, a determination ofpolyploidy is made by determining if there are more than 2 signals fromthe gene of interest. In some embodiments, polyploidy is determined bymeasuring the signal from the probe specific for the gene of interestand comparing it with a centromeric probe or other probe.

Method of Prognosis of Clinical Outcome in Lung Cancer Based onDefecting the Amplification or Translocation of the c-MAF Gene

In another aspect, the invention relates to an in vitro method(hereinafter seventh method of the invention) for predicting theclinical outcome of a patient suffering lung cancer, which comprisesdetermining if the c-MAF gene is amplified or translocated in a sampleof said subject relative to a reference gene copy number wherein anamplification of the c-MAF gene with respect to said reference gene copynumber is indicative of a poor clinical outcome.

The seventh method of the invention comprises in a first step,determining if the c-MAF gene is amplified in a sample of a subject. Thedetermination of the amplification of the c-MAF is carried outessentially as described in the fifth method of the invention. In apreferred embodiment the sample is a tumor tissue sample. In a preferredembodiment, the amplification of the c-MAF gene is determined by meansof determining the amplification of the locus 16q23 or 16q22-q24. Inanother preferred embodiment, the amplification of the c-MAF gene isdetermined by means of using a c-MAF gene-specific probe.

In a second step, the seventh method of the invention comprisescomparing said copy number with the copy number of a control orreference sample, wherein if the c-MAF copy number is greater withrespect to the c-MAF copy number of a control sample, then this isindicative of a poor clinical outcome.

In a preferred embodiment the c-MAF gene is amplified with respect to areference gene copy number when the c-MAF gene copy number is higherthan the copy number that a reference sample or control sample has. Inone example, the c-MAF gene is said to be “amplified” if the genomiccopy number of the c-MAF gene is increased by at least 2- (i.e., 6copies), 3- (i.e., 8copies), 4-, 5-, 6-, 7-, 8-, 9-, 10-, 15-, 20-, 25-,30-, 35-, 40-, 45-, or 50-fold in a test sample relative to a controlsample. In another example, a c-MAF gene is said to be “amplified” ifthe genomic copy number of the c-MAF gene per cell is at least 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, and the like.

In another embodiment, the reference gene copy number is the gene copynumber in a sample of lung cancer, from a subject who has not sufferedbone metastasis.

In another embodiment, the amplification is determined by means of insitu hybridization or PCR.

Therapeutic Methods of the Invention

Treating bone metastasis using c-MAF inhibitory agents.

A c-MAF gene expression inhibitory agent or an inhibitory agent of theprotein encoded by said gene can be used in the treatment and/or theprevention of lung cancer metastasis.

In another aspect, the invention relates to the use of a c-MAFinhibitory agent for the manufacture of a medicament for the treatmentor prevention of bone metastasis lung cancer.

In another aspect, the invention relates to a method for the treatmentor prevention of the bone metastasis from lung cancer, in a subject inneed thereof comprising the administration to said subject of a c-MAFinhibitory agent.

In another aspect, the invention relates to a method for preventing orreducing the risk of bone metastasis in a subject suffering from lungcancer, said method comprising administering to said subject an agentthat prevents or reduces bone metastasis, wherein said agent isadministered in accordance with a treatment regimen determined fromquantifying the expression level of c-MAF in said subject.

Therefore, in another aspect, the invention relates to the use of ac-MAF gene expression inhibitory agent or an inhibitory agent of theprotein encoded by said gene (hereinafter, inhibitory agent of theinvention) in the preparation of a medicinal product for treating and/orpreventing lung cancer metastasis. Alternatively, the invention relatesto a c-MAF gene expression inhibitory agent or an inhibitory agent ofthe protein encoded by said gene for use in the treatment and/or theprevention of lung cancer metastasis. Alternatively, the inventionrelates to a method for treating the lung cancer metastasis in a subjectwhich comprises administering a c-MAF inhibitor to said subject.

As used herein, a “c-MAF inhibitory agent” refers to any moleculecapable of completely or partially inhibiting the c-MAF gene expression,both by preventing the expression product of said gene from beingproduced (interrupting the c-MAF gene transcription and/or blocking thetranslation of the mRNA coming from the c-MAF gene expression) and bydirectly inhibiting the c-MAF protein activity. C-MAF gene expressioninhibitors can be identified using methods based on the capacity of theso-called inhibitor to block the capacity of c-MAF to promote the invitro cell proliferation, such as shown in the international patentapplication WO2005/046731 (hereby incorporated by reference in itsentirety), based on the capacity of the so-called inhibitor to block thetranscription capacity of a reporter gene under the control of thecyclin D2 promoter or of a promoter containing the c-MAF response region(MARE or c-MAF responsive element) in cells which express c-MAF such asdescribed in WO2008098351 (hereby incorporated by reference in itsentirety) or based on the capacity of the so-called inhibitor to blockthe expression of a reporter gene under the control of the IL-4 promoterin response to the stimulation with PMA/ionomycin in cells which expressNFATc2 and c-MAF such as described in US2009048117A (hereby incorporatedby reference in its entirety).

By way of non-limiting illustration, c-MAF inhibitory agents suitablefor use in the present invention include antisense oligonucleotides,interference RNAs (siRNAs), catalytic RNAs or specific ribozymes andinhibitory antibodies.

Antisense Oligonucleotides

An additional aspect of the invention relates to the use of isolated“antisense” nucleic acids to inhibit expression, for example, forinhibiting transcription and/or translation of a nucleic acid whichencodes c-MAF the activity of which is to be inhibited. The antisensenucleic acids can be bound to the target potential of the drug by meansof conventional base complementarity or, for example, in the case ofbiding to Double stranded DNA through specific interaction in the largegroove of the double helix. Generally, these methods refer to a range oftechniques generally used in the art and they include any method whichis based on the specific binding to oligonucleotide sequences.

An antisense construct of the present invention can be administered, forexample, as an expression plasmid which, when is transcribed in cell,produces RNA complementary to at least one unique part of the cellularmRNA encoding c-MAF. Alternatively, the antisense construct is aoligonucleotide probe generated ex vivo which, when introduced into thecell, produces inhibition of gene expression hybridizing with the mRNAand/or gene sequences of a target nucleic acid. Such oligonucleotideprobes are preferably modified oligonucleotides which are resistant toendogenous nucleases, for example, exonucleases and/or endonucleases andare therefore stable in vivo. Examples of nucleic acids molecules foruse thereof as an antisense oligonucleotides are DNA analogs ofphosphoramidate, phosphothionate and methylphosphonate (sec also U.S.Pat. Nos. 5,176,996; 5,264,564; and 5,256,775) (hereby incorporated byreference in their entireties). Additionally, the general approximationsfor constructing oligomers useful in the antisense therapy have beenreviewed, for example, in Van der Krol et al., BioTechniques 6: 958-976,1988; and Stein et al. Cancer Res 48:2659-2668, 1988.

With respect to the antisense oligonucleotide, theoligodeoxyribonucleotide regions derived from the starting site of thetranslation, for example, between −10 and +10 of the target gene arepreferred. The antisense approximations involve the oligonucleotidedesign (either DNA or RNA) that are complementary to the mRNA encodingthe target polypeptide. The antisense oligonucleotide will be bound tothe transcribed mRNA and translation will be prevented.

The oligonucleotides which are complementary to the 5′ end of the mRNA,for example the non translated 5′ sequence up to and including the startcodon AUG must function in the most efficient manner to inhibittranslation. Nevertheless, it has been shown that the sequencescomplementary to the non translated 3′ sequences of the mRNA are alsoefficient for inhibiting mRNA translation (Wagner, Nature 372:333,1994). Therefore, complementary oligonucleotides could be used at thenon translated 5′ or 3′ regions, non coding regions of a gene in anantisense approximation to inhibit the translation of that mRNA. Theoligonucleotides complementary to the non translated 5′ region of themRNA must include the complement of the start codon AUG. Theoligonucleotides complementary to the coding region of the mRNA are lessefficient translation inhibitors but they could also be used accordingto the invention. If they are designed to hybridize with the 5′ region,3′ region or the coding region of the mRNA, the antisense nucleic acidsmust have at least six nucleotides long and preferably have less thanapproximately 100 and more preferably less than approximately 50, 25, 17or 10 nucleotides long.

Preferably, in vitro studies are performed first to quantify thecapacity of the antisense oligonucleotides for inhibiting geneexpression. Preferably, these studies use controls which distinguishbetween antisense gene inhibition and nonspecific biological effects ofthe oligonucleotides. Also preferably these studies compared the levelsof target RNA or protein with that of an internal control of RNA orprotein. The results obtained using the antisense oligonucleotides canbe compared with those obtained using a control oligonucleotide.Preferably the control oligonucleotide is approximately of the samelength as the oligonucleotide to be assayed and that the oligonucleotidesequence does not differ from the antisense sequence more than it isdeemed necessary to prevent the specific hybridization to the targetsequence.

The antisense oligonucleotide can be a single or double stranded DNA orRNA or chimeric mixtures or derivatives or modified versions thereof.The oligonucleotide can be modified in the base group, the sugar groupor the phosphate backbone, for example, to improve the stability of themolecule, its hybridization capacity etc. The oligonucleotide mayinclude other bound groups, such as peptides (for example, for directingthem to the receptors of the host cells) or agents for facilitatingtransport through the cell membrane (see, for example, Letsinger et al.,Proc. Natl. Acad. Sci. U.S.A. 86:6553-6556, 1989, Lemaitre et al., Proc.Natl. Acad. Sci. 84:648-652, 1987; PCT Publication No. WO 88/09810) orthe blood-brain barrier (see, for example, PCT Publication No. WO89/10134), intercalating agents, (see, for example, Zon, Pharm. Res.5:539-549, 1988). For this purpose, the oligonucleotide can beconjugated to another molecule, for example, a peptide, a transportingagent, hybridization triggered cleaving agent, etc.

The antisense oligonucleotides may comprise at least one group ofmodified base. The antisense oligonucleotide may also comprise at leasta modified sugar group selected from the group including but not limitedto arabinose, 2-fluoroarabinose, xylulose, and hexose. The antisenseoligonucleotide may also contain a backbone similar to a neutralpeptide. Such molecules are known as peptide nucleic acid (PNA)oligomers and are described, for example, in Perry-O'Keefe et al., Proc.Natl. Acad. Sci. U.S.A. 93:14670, 1996, and in Eglom et al., Nature365:566, 1993.

In yet another embodiment, the antisense oligonucleotide comprises atleast one modified phosphate backbone. In yet another embodiment, theantisense oligonucleotide is an alpha-anomeric oligonucleotide.

While antisense oligonucleotides complementary to the coding region ofthe target mRNA sequence can be used, those complementary to thetranscribed non translated region can also be used.

In some cases, it may be difficult to reach the sufficient intracellularconcentrations of the antisense to suppress the endogenous mRNAtranslation. Therefore, a preferred approximation uses a recombinant DNAconstruct in which the antisense oligonucleotide is placed under thecontrol of a strong pol III or pol II promoter.

Alternatively, the target gene expression can be reduced by directingdeoxyribonucleotide sequences complementary to the gene regulatingregion (i.e., the promoter and/or enhancers) to form triple helixstructures preventing gene transcription in the target cells in the body(see in general, Helene, Anticancer Drug Des. 6(6): 569-84, 1991). Incertain embodiments, the antisense oligonucleotides are antisensemorpholines.

siRNA

Small interfering RNA or siRNA are agents which are capable ofinhibiting the expression of a target gene by means of RNA interference.A siRNA can be chemically synthesized, can be obtained by means of invitro transcription or can be synthesized in vivo in the target cell.Typically, the siRNA consist of a double stranded RNA between 15 and 40nucleotide long and may contain a 3′ and/or 5′ protruding region of 1 to6 nucleotides. The length of the protruding region is independent of thetotal length of the siRNA molecule. The siRNA act by means of degradingor silencing the target messenger after transcription.

The siRNA of the invention are substantially homologous to the mRNA ofthe c-MAF encoding gene or to the gene sequence which encodes saidprotein. “Substantially homologous” is understood as having a sequencewhich is sufficiently complementary or similar to the target mRNA suchthat the siRNA is capable of degrading the latter through RNAinterference. The siRNA suitable for causing said interference includesiRNA formed by RNA, as well as siRNA containing different chemicalmodifications such as

-   -   siRNA in which the bonds between the nucleotides are different        than those appear in nature, such as phosphorothionate bonds.    -   Conjugates of the RNA strand with a functional reagent, such as        a fluorophore.    -   Modifications of the ends of the RNA strands, particularly of        the 3′ end by means of the modification with different hydroxyl        functional groups in 2′ position.    -   Nucleotides with modified sugars such as O-alkylated residues on        2′ position like 2′-O-Nucleotides or 2′-O-fluororibose.    -   Nucleotides with modified bases such as halogenated bases (for        example 5-bromouracil and 5-iodouracil), alkylated bases (for        example 7-methylguanosine).

The siRNA can be used as is, i.e., in the form of a double stranded RNAwith the aforementioned characteristics. Alternatively, the use ofvectors containing the sense and antisense strand sequence of the siRNAis possible under the control of suitable promoters for the expressionthereof in the cell of interest.

Vectors suitable for expressing siRNA are those in which the two DNAregions encoding the two strands of siRNA are arranged in tandem in oneand the same DNA strand separated by a spacer region which, upontranscription, forms a loop and wherein a single promoter directs thetranscription of the DNA molecule giving rise to shRNA.

Alternatively, the use of vectors in which each of the strands formingthe siRNA is formed from the transcription of a differenttranscriptional unit is possible. These vectors are in turn divided intodivergent and convergent transcription vectors. In divergenttranscription vectors, the transcriptional units encoding each of theDNA strands forming the siRNA are located in tandem in a vector suchthat the transcription of each DNA strand depends on its own promoterwhich may be the same or different (Wang, J. et al., 2003, Proc. Natl.Acad. Sci. USA., 100:5103-5106 and Lee, N. S., et al., 2002, Nat.Biotechnol., 20:506-505). In convergent transcription vectors, the DNAregions giving rise to the siRNA form the sense and antisense strands ofa DNA region which are flanked by two reverse promoters. After thetranscription of the sense and antisense RNA strands, the latter willform the hybrid for forming a functional siRNA. Vectors with reversepromoter systems in which 2 U6 promoters (Tran, N. et al., 2003, BMCBiotechnol., 3:21), a mouse U6 promoter and a human H1 promoter (Zheng,L., et al., 2004, Proc. Natl. Acad. Sci. USA., 135-140 and WO2005026322) and a human U6 promoter and a mouse H1 promoter (Kaykas, A.and Moon, R., 2004, BMC Cell Biol., 5:16) are used have been described.

Promoters suitable for use thereof in the expression of siRNA fromconvergent or divergent expression vectors include any promoter, or pairof promoters compatible with the cells in which the siRNA is to beexpressed. Thus, promoters suitable for the present invention includebut are not necessarily limited to constitutive promoters such as thosederived from the genomes of eukaryotic viruses such as the polyomavirus, adenovirus, SV40, CMV, avian sarcoma virus, hepatitis B virus,the metallothionein gene promoter, the thymidine kinase gene promoter ofthe herpes simplex virus, retrovirus LTR regions, the immunoglobulingene promoter, the actin gene promoter, the EF-1alpha gene promoter aswell as inducible promoters in which the protein expression depends onthe addition of a molecule or an exogenous signal such as thetetracycline system, the NFkappaB/UV light system, the Cre/Lox systemand the heat shock gene promoter, the regulatable RNA polymerase IIpromoters described in WO/2006/135436 as well as specific tissuepromoters (for example, the PSA promoter described in WO2006012221). Ina preferred embodiment, the promoters are RNA polymerase III promoterswhich act constitutively. The RNA polymerase III promoters are found ina limited number of genes such as 5S RNA, tRNA, 7SL RNA and U6 snRNA.Unlike other RNA polymerase III promoters, type III prompters do notrequire any intragenic sequence but rather need sequences in 5′direction comprising a TATA box in positions −34 and −24, a proximalsequence element or DSE between −66 and −47 and, in some cases, a distalsequence element or DSE between positions −265 and −149. In a preferredembodiment, the type III RNA polymerase III promoters are the human ormurine H1 and U6 gene promoters. In a yet more preferred embodiment, thepromoters are 2 human or murine U6 promoters, a mouse U6 promoter and ahuman H1 promoter or a human U6 promoter and a mouse H1 promoter.

The siRNA can be generated intracellularly from the so called shRNA(short hairpin RNA) characterized in that the antiparallel strandsforming the siRNA are connected by a loop or hairpin region. The shRNAscan be encoded by plasmids or viruses, particularly retroviruses, andare under the control of a promoter. Promoters suitable for expressingshRNA are those indicated in the paragraph above for expressing siRNA.

Vectors suitable for expressing siRNA and shRNA include prokaryoticexpression vectors such as pUC18, pUC19, Bluescript and the derivativesthereof, mp18, mp19, pBR322, pMB9, ColE1, pCR1, RP4, phages and shuttlevectors such as pSA3 and pAT28, yeast expression vectors such as2-micron plasmid type vectors, integration plasmids, YEP vectors,centromeric plasmids and the like, insect cell expression vectors suchas pAC series vectors and pVL series vectors, plant expression vectorssuch as pIBI, pEarleyGate, pAVA, pCAMBlA, pGSA, pGWB, pMDC, pMY, pOREseries vectors and the like and viral vector-based (adenovirus, virusesassociated with adenoviruses as well as retroviruses and particularlylentiviruses) higher eukaryotic cell expression vectors or non-viralvectors such as pcDNA3, pHCMV/Zeo, pCR3.1, pEF1/His, pIND/GS, pRc/HCMV2,pSV40/Zeo2, pTRACER-HCMV, pUB6/V5-His, pVAX1, pZeoSV2, pCI,, pSVL andpKSV-10, pBPV-1, pML2d and pTDT1. In a preferred embodiment, the vectorsare lentiviral vectors.

The siRNA and shRNA of the invention can be obtained using a series oftechniques known by the person skilled in the art. The region of thenucleotide sequence taken as a basis for designing the siRNA is notlimiting and it may contain a region of the coding sequence (between thestart codon and the end codon) or it may alternatively contain sequencesof the non-translated 5′ or 3′ region preferably between 25 and 50nucleotides long and in any position in 3′ direction position withrespect to the start codon. One way of designing an siRNA involves theidentification of the AA(N19)TT motifs wherein N can be any nucleotidein the c-MAF gene sequence, and the selection of those having a high G/Ccontent. If said motif is not found, it is possible to identify theNA(N21) motif wherein N can be any nucleotide.

c-MAF specific siRNAs include the siRNA described in WO2005046731(hereby incorporated by reference in its entirety), one of the strandsof which is ACGGCUCGAGCAGCGACAA (SEQ ID NO: 6). Other c-MAF specificsiRNA sequences include but are not limited to CUUACCAGUGUGUUCACAA (SEQID NO: 7), UGGAAGACUACUACUGGAUG (SEQ ID NO: 8), AUUUGCAGUCAUGGAGAACC(SEQ ID NO: 9), CAAGGAGAAAUAGGAGAAGU (SEQ ID NO: 10),ACAAGGAGAAAUACGAGAAG (SEQ ID NO: 11) and ACCUGGAAGAGUACUACUGG (SEQ IDNO: 12).

DNA Enzymes

On the other hand, the invention also contemplates the use of DNAenzymes to inhibit the expression of the c-MAF gene of the invention.DNA enzymes incorporate some of the mechanistic features of bothantisense and ribozyme technologies. DNA enzymes are designed such thatthey recognize a particular target nucleic acid sequence similar to theantisense oligonucleotide, nevertheless like the ribozyme they arecatalytic and specifically cleave the target nucleic acid.

Ribozymes

Ribozyme molecules designed for catalytically cleaving transcriptionproducts of a target mRNA to prevent the translation of the mRNA whichencodes c-MAF the activity of which is to be inhibited, can also beused. Ribozymes are enzymatic RNA molecules capable of catalysingspecific RNA cleaving. (For a review, see, Rossi, Current Biology4:469-471, 1994). The mechanism of ribozyme action involves a Specifichybridization of a ribozyme molecule sequence to a complementary targetRNA followed by an endonucleolytic cleavage event. The composition ofthe ribozyme molecules preferably includes one or more sequencescomplementary to the target mRNA and the well known sequence responsiblefor cleaving the mRNA or a functionally equivalent sequence (see, forexample, U.S. Pat. No. 5,093,246).

The ribozymes used in the present invention include hammer-headribozymes, endoribonuclease RNA (hereinafter “Cech type ribozymes”)(Zaug et. al., Science 224:574-578, 1984.

The ribozymes can be formed by modified oligonucleotides (for example toimprove the stability, targeting, etc.) and they should be distributedto cells expressing the target gene in vivo. A preferred distributionmethod involves using a DNA construct which “encodes” the ribozyme underthe control of a strong constitutive pol III or pol II promoter suchthat the transfected cells will produce sufficient amounts of theribozyme to destroy the endogenous target messengers and to inhibittranslation. Since the ribozymes are catalytic, unlike other antisensemolecules, a low intracellular concentration is required for itsefficiency.

Inhibitory Antibodies

In the context of the present invention, “inhibitory antibody” isunderstood as any antibody capable of binding specifically to the c-MAFprotein and inhibiting one or more of the functions of said protein,preferably those related to transcription. The antibodies can beprepared using any of the methods which are known by the person skilledin the art, some of which have been mentioned above. Thus, thepolyclonal antibodies are prepared by means of immunizing an animal withthe protein to be inhibited. The monoclonal antibodies are preparedusing the method described by Kohler, Milstein et al. (Nature, 1975,256: 495). In the context of the present invention, suitable antibodiesinclude intact antibodies comprising a variable antigen binding regionand a constant region, “Fab”, “F(ab′)2” and “Fab′”, Fv, scFv fragments,diabodies, bispecific antibodies, alphabodies, cyclopeptides and stapledpeptides. Once antibodies with c-MAF protein binding capacity areidentified, those capable of inhibiting the activity of this proteinwill be selected using an inhibitory agent identification assay.

Inhibitory Peptides

As used herein, the term “inhibitory peptide” refers to those peptidescapable of binding to the c-MAF protein and inhibiting its activity ashas been explained above, i.e., preventing the c-MAF from being able toactivate gene transcription.

Negative c-MAF Dominants

Since the proteins from the maf family are capable of homodimerizing andheterodimerizing with other members of the AP-1 family such as Fos andJun, one way of inhibiting c-MAF activity is by means of using negativedominants capable of dimerizing with c-MAF but lacking the capacity foractivating transcription. Thus, the negative c-MAF dominants can be anyof the small maf proteins existing in the cell and lacking two-thirds ofthe amino terminal end containing the transactivation domain (forexample, mafK, mafF, mafg and pi 8) (Fujiwara et al (1993) Oncogene 8,2371-2380, Igarashi et al. (1995) J. Biol. Chem. 270, 7615-7624; Andrewset al. (1993) Proc. Natl. Acad. Sci. USA 90, 11488-11492, Kataoka et al.(1995) Mol. Cell. Biol. 15, 2180-2190) (Kataoka et al. (1996) Oncogene12, 53-62).

Alternatively, the negative c-MAF dominants include c-MAF variants whichmaintain the capacity for dimerizing with other proteins but lack thecapacity for activating transcription. These variants are, for example,those lacking the c-MAF transactivation domain located at the N-terminalend of the protein. Thus, negative c-MAF dominant variants include in anillustrative manner the variants in which at least amino acids 1 to 122,at least amino acids 1-187 or at least amino acids 1 to 257 (byconsidering the numbering of human c-MAF as described in U.S. Pat. No.6,274,338, hereby incorporated by reference in its entirety) have beenremoved.

The invention contemplates the use of both the negative c-MAF dominantvariants and of polynucleotides encoding c-MAF under the operativecontrol of a promoter suitable for expression in target cell. Thepromoters that can be used for regulating the polynucleotidetranscription of the invention can be constitutive promoters, i.e.,promoters directing the transcription at a basal level, or induciblepromoters in which the transcriptional activity requires an externalsignal. Constitutive promoters suitable for regulating transcriptionare, among others, the CMV promoter, the SV40 promoter, the DHFRpromoter, the mouse mammary tumor virus (MMTV) promoter, the 1aelongation factor (EF1a) promoter, the albumin-promoter, the ApoA1promoter, the keratin promoter, the CD3 promoter, the immunoglobulinheavy or light chain promoter, the neurofilament promoter, the neuronspecific enolase promoter, the L7 promoter, the CD2 promoter, the myosinlight chain kinase promoter, the HOX gene promoter, the thymidine kinasepromoter, the RNA polymerase II promoter, the MyoD gene promoter, thephosphoglyceratekinase (PGK) gene promoter, the low density lipoprotein(LDL) promoter, the actin gene promoter. In a preferred embodiment, thepromoter regulating the expression of the transactivator is the PGK genepromoter. In a preferred embodiment, the promoter regulating thepolynucleotide transcription of the invention is the RNA polymerasepromoter of the T7 phage.

Preferably, the inducible promoters that can be used in the context ofthe present invention are those responding to an inducer agent showingzero or negligible basal expression in the absence of an inducer agentand are capable of promoting the activation of gene located in the 3′position. Depending on the type of inducer agent, the induciblepromoters are classified as Tet on/off promoters (Gossen, M. and H.Bujard (1992) Proc. Natl. Acad. Sci. USA, 89:5547-5551: Gossen, M. etal., 1995, Science 268:1766-1769; Rossi, F. M. V. and H. M. Blau, 1998,Curr. Opin. Biotechnol. 9:451-456); Pip on/off promoters (U.S. Pat. No.6,287,813); antiprogestin-dependent promoters (US 2004132086),ecdysone-dependent promoters (Christopherson et al., 1992, Proc. Natl.Acad. Sci. USA, 89:6314-6318; No et al., 1996; Proc. Natl. Acad. Sci.USA, 93:3346-3351, Suhr et al., 1998, Proc. Natl. Acad. Sci. USA,95:7999-8004 and WO9738117), a metallothionein-dependent promoter(WO8604920) and rapamycin-dependent promoters (Rivera et al., 1996, Nat.Med. 2:1028-32).

Vectors suitable for expressing the polynucleotide encoding the negativec-MAF dominant variant include vectors derived from prokaryoticexpression; vectors such, as pUC18, pUC19, Bluescript and derivativesthereof, mp18, mp19, pBR322, pMB9, ColE1, pCR1, RP4, phages and shuttlevectors such as pSA3 and pAT28, yeast expression vectors such as2-micron type plasmid vectors, integration plasmids, YEP vectorscentromeric plasmids and the like, insect cell expression vectors suchas pAC series vectors and pVL series vectors, plant expression vectorssuch as pIBI, pEarleyGate, pAVA, pCAMBIA, pGSA, pGWB, pMDC, pMY, pOREseries vectors and the like and viral vector-based (adenoviruses,viruses associated with adenoviruses as well as retroviruses andparticularly lentiviruses) higher eukaryotic cell expression vectors ORnon-viral vectors such as pSilencer 4.1-CMV (Ambion), pcDNA3,pcDNA3.1/hyg pHCMV/Zeo, pCR3.1, pEF1/His, pIND/GS, pRc/HCMV2,pSV40/Zeo2, pTRACER-HCMV, pUB6/V5-His, pVAX1, pZeoSV2, pCI, pSVL andpKSV-10; pBPV-1, pML2d and pTDTI.

Other Inhibitory Compounds of the c-MAF Protein Activity

Other c-MAF inhibitory compounds suitable for use in the presentinvention include:

TABLE 1 small molecules with c-MAF inhibiting capacity I Endiandric acidH derivatives such as those described in WO2004014888 corresponding tothe general formula

wherein R₁ and R₂ are, independently of one another, 1.0 H or 2.0 aO—C₁-C₆-alkyl, —O—C₂-C₆-alkenyl, —O—C₂-C₆-alkynyl or —O—C₆-C₁₀-arylgroup, in which alkyl, alkenyl and alkynyl are straight-chain orbranched, and in which the alkyl, alkenyl and alkynyl groups are mono-or disubstituted with: 2.1 —OH, 2.2 ═O, 2.3 —O—C₁-C6-alkyl, in whichalkyl is straight-chain or branched, 2.4 —O—C₂-C₆-alkenyl, in whichalkenyl is straight-chain or branched, 2.5 C₆-C₁₀-aryl, 2.6—NH—C₁-C₆-alkyl, in which alkyl is straight-chain or branched, 2.7—NH—C₂-C₆-alkenyl, in which alkenyl is straight-chain or branched, 2.8—NH₂ or 2.9 halogen, and in which the aryl group, is optionally mono- ordisubstituted with the substituent 2.1 or 2.3 to 2.9, in which thesubstituents 2.3, 2.4, 2.6 and 2.7 may be further substituted with —CN,-amide or -oxime functions, and 2.5 may be further substituted with —CNor amide functions, or R₁ and R₂ together form a ring, wherein R₁ and R₂mean a —O—[(C₁-C₆)-alkylene]-O— group, R₃ is 1.0 H or 2.0 a—O—C₁-C₆-alkyl, —O—C₂-C₆-alkenyl, —O—C₂-C₆-alkynyl or —O—C₆-C₁₀-arylgroup, in which alkyl, alkenyl and alkynyl are straight-chain orbranched, and in which the alkyl, alkenyl and alkynyl groups are mono-or disubstituted with: 2.1 —OH, 2.2 ═O, 2.3 —O—C₁-C₆-alkyl, in whichalkyl is straight-chain or blanched, 2.4 —O—C₂-C₆-alkenyl, in whichalkenyl is straight-chain or branched, 2.5 —C₆-C₁₀-aryl, 2.6—NH—C₁-C₆-alkyl, in which alkyl is straight-chain or branched, 2.7—NH—C₂-C₆-alkenyl, in which alkenyl is straight-chain or branched, 2.8—NH₂ or 2.9 halogen, and in which the aryl group, is optionally mono- ordisubstituted with the substituent 2.1 or 2.3 to 2.9, in which thesubstituents 2.3, 2.4, 2.6 and 2.7 may be further substituted with —CN,-amide or -oxime functions, and 2.5 may be further substituted with —CNor amide functions R₄ is CO₂R₃, CO₂NHR₃, CHO, CH₂OR₃, CH₂OSi(R₃)₃,CH₂Br, CH₂CN, in which R₃ is as defined above, and, in particular, thecompounds

II 8-hydroxyquinoline derivatives such as those described inWO2009146546 of general formula

wherein R₁ is selected from the group consisting of NO₂, NH₂,NH(C₁-C₆-alkyl) and N(C₁-C₆-alkyl)(C₁-C₆-alkyl); R₂ is selected from H,halogen, C₁-C₆ alkyl, and fluoro-substituted C₁-C₆ alkyl, or R₁ is Cland R₂ is Br or H, and, preferably, the compounds

III Clioquinol (5-chloro-7-iodoquinolin-8-ol) as described in WO09049410IV Compounds such as those described in WO08098351 of general formula

wherein ==-:-:-: is a single or double bond, R¹ is selected from thegroup consisting of H, C₁-C₄ alkyl, C(O)O C₁-C₄ alkyl, C(O) C₁-C₄ alkyland C(O)NH C₁-C₄ alkyl; R² is selected from H and C₁-C₄ alkyl; R³ isselected from H and C₁-C₄ alkyl; or R² and R³ are bound together alongwith the carbon and nitrogen atoms to which they are bound to form apiperidine ring, R⁴ and R⁵ are independently selected from H, halogen,hydroxy, C₁-C₄ alkyl, fluoro-substituted C₁-C₄ alkyl and C₁-C₄ alkoxy;and X is selected from C and N, and preferred compounds such asCyproheptadine (4-(5H-dibenzo-[a,d]cyclohepten-5-ylidene)-1-methylpiperidine hydrochloride), Amitriptyline(3-(10,11-dihydro-5H-dibenzo[[a,d]]cycloheptene-5-ylidene)-N,N-dimethyl-1-propanamine), Loratadine (Ethyl4-(8-chloro-5,6-dihydro-11H-benzo[5,6]cyclohepta[1,2-b]pyridin-11-ylidene)-1-piperidinecarboxylate,Cyclobenzrapine (3-(5H-dibenzo[a,d]cyclohepten-5-ylidene)-N,N-dimethyl-1-propanamine). V Nivalenol(12,13-epoxy-3,4,7,15-tetrahydroxytrichothec-9-en-8- one) as describedin WO0359249

Other c-MAF inhibitors are described in the patent applicationWO2005063252, such as shown in the following table (Table 2).

TABLE 2 c-MAF inhibitors Antagonist Reference for cdk2 inhibitoryactivity Purine Analogs Purvalanols such as 2-(1R-Isopropyl-2- Gray, N.S. et al., Science, 281, 533-538hydroxyethylamino)-6-(3-chloroanilino)-9- (1998); isopropylpurine havinga molecular formula Chang, Y. T. et al., Chem. Biol., 6, 361-375C₁₉H₂₅ClN₆O available from Sigma-Aldrich under (1999). the trade namePurvalanol A (#P4484, Sigma- Aldrich, St. Louis, MO), Purvalanol B,aminopurvalanol, compound 52 (where isopropyl of purvalanol A isreplaced with H) 2-(Hydroxyethylamino)-6-benzylamino-9- Vesely, J., etal., (1994) Eur, J. Biochem., 224, methylpurine having a molecularformula 771-86, 11; C₁₅H₁₈N₆O available from Sigma-Aldrich under Brooks,E. E., et al., (1997) J. Biol. Chem., 272, the trade name Olomoucine(#O0886), 29207-11 2-(2′-Hydroxyethylamino)-6-benzylamino-9-isopropylpurine having a molecular formula C₁₇H₂₂N₆O available fromSigma-Aldrich under the trade name N⁹-isopropylolomoucine (#I0763);CVT-313 6-(Benzylamino)-2(R)-[[1- Wang, D. et al., J. Virol., 75,7266-7279 (hydroxymethyl)propyl]amino]-9-isopropylpurine (2001); McClue,S., et al., Int. J. Cancer, 102, 2-(R)-[[9-(1-methylethyl)-6- 463-468(2002); [(phenylmethyl)amino]-9H-purin-2-yl]amino]-1- Meijer, L., etal., (1997) Eur. J. Biochem., 243, butanol having a molecular formula ofC₁₉H₂₆N₆O 527-36 available from Sigma-Aldrich under the trade nameRoscovitine (#R7772), methoxyroscovitine Purine analogN2-(cis-2-Aminocyclohexyl)-N6- Imbach, P. et al., Bioorg. Med. Chem.Lett., 9, (3-chlorophenyl)-9-ethyl-9H-purine-2,6-diamine 91-96 (1999);having a molecular formula of C₁₉H₂₄ClN₇ Dreyer, M. K. et al., J. Med.Chem., 44, 524-530 available from Sigma-Aldrich under the trade (2001).name CGP74514 (#C3353) CGP79807, a purine analog of CGP74514 (supra)Imbach, P. et al., Bioorg. Med. Chem. Lett., 9, where Cl is replacedwith CN, OH is removed, 91-96 (1999); and the ortho position ofcyclohexane ring is NH₂ Dreyer, M. K. et al., J. Med. Chem., 44, 524-530(2001). purine analog such as O6-cyclohexylmethyl Arris, C. E. et al.,J. Med. Chem., 43, 2797-2804 guanine NU2058 (2000); Davies et al, NatureStructural Biology, 9: 10, 745-749, 2002 purine analog such as NU6102Arris, C. E. et al., J. Med. Chem., 43, 2797-2804 (2000); Davies, T. G.et al., Nat. Struct. Biol., 9, 745-749 (2002). isopentenyl-adenineVesely, J., et al., (1994) Eur. J. Biochem., 224, 771-86 Nonpurine basedagents Indirubins such as indirubin-3′-monoxime having Davies, T. G. etal., Structure, 9, 389-397 a molecular formula of C₁₆H₁₁N₃O₂ availablefrom (2001); Sigma-Aldrich under the trade name (#I0404), Marko, D. etal., Br. J. Cancer, 84, 283-289 indirubin 5-sulfonate, 5-chloroindirubin (2001); Hoessel, R., et al., (1999) Nat. Cell Biol., 1, 60-7;PCT/US02/30059 to Hellberg et al., published as WO 03/027275. Oxindole 1of Fischer as referenced in column 2 Porcs-Makkay, M., et al.,Tetrahedron 2000, of this table, (#IN118, JMAR Chemical, 56, 5893; Org.Process Res. Dev. 2000, 4, 10 Indenopyrazoles Nugiel, D. A. et al., J.Med. Chem., 44, 1334-1336 (2001); Nugiel, D. A. et al., J. Med. Chem.,45, 5224-5232 (2002); Yue, E. W. et al., J. Med. Chem., 45, 5233-5248(2002). Pyrido(2,3-d)pyrimidine-7-ones, compound 3 of Barvian, M. etal., J. Med. Chem., 43, 4606-4616 Fischer (2000); Toogood, P. L., Med.Res. Rev., 21, 487-498 (2001). Quinazolines such as anilinoquinazolineSielecki, T. M. et al., Bioorg. Med. Chem. Lett., 11, 1157-1160 (2001);Mettey et al., J. Med. Chem. 2003, 46, 222-236. Thiazoles such as fusedthiazole, 4-{[(7-Oxo-6,7- Davis, S. T. et al., Science, 291, 134-137dihydro-8H-[1,3]thiazolo[5,4-e]indol-8- (2001);ylidene)methyl]amino}-N-(2- PCT/US02/30059 to Hellberg et al., publishedpyridyl)benzenesulfonamide having a molecular as WO 03/027275. formulaof C₂₁H₁₅N₅O₃S₂ available from Sigma- Aldrich under the trade nameGW8510 (#G7791) Flavopiridols such as flavopiridol (L86 8275; Carlson,B. A., et al., (1996) Cancer Res., 56, NCS 649890, National CancerInstitute, Bethesda, 2973-8 MD) and a dechloro derivative Alkaloids suchas Staurosporine (#S1016, A.G. Rislet, V., et al., (1991) AnticancerRes., 11, Scientific, San Diego, CA) or UCN-01 (7- 1581-90;hydroxystaurosporine) National Cancer Institute, Wang, Q., et al.,(1995) Cell Growth Differ., 6, Bethesda, MD 927-36, Akiyama, T., et al.,(1997) Cancer Res., 57, 1495-501, Kawakami, K., et al., (1996) Biochem.Biophys. Res. Commun., 219, 778-83 Paullones such as9-Bromo-7,12-dihydro- Zaharevitz, D. W. et al., Cancer Res., 59,2566-2569 indolo[3,2-d][1]benzazepin-6(5H)-one having a (1999); Schultz,C. et al., J. Med. Chem., molecular formula of C₁₆H₁₁BrN₂O availablefrom 42, 2909-2919 (1999); Sigma-Aldrich under the trade namekenpaullone Zaharevitz, D. W., et al., (1999) Cancer Res., (#K3888), or9-Nitro-7,12-dihydroindolo-[3,2- 59, 2566-9; d][1]benzazepin-6(5)-onehaving a molecular PCT/US02/30059 to Hellberg et al., published formulaof C₁₆H₁₁N₃O₃ available from Sigma- as WO 03/027275. Aldrich under thetrade name alsterpaullone (#A4847) CGP 41251, an alkaloid Begemann, M.,et al., (1998) Anticancer Res., 18, 2275-82; Fabbro et al., PharmacolTher. 1999 May-June; 82(2-3): 293-301 Hymenialdisines such as10z-hymenialdisine Meijer, L., et al., (1999) Chemistry & Biology,having a molecular formula of C₁₁H₁₀BrN₅O₂ 7, 51-63; available fromBiochemicals.net, a division of PCT/US02/30059 to Hellberg et al.,published A.G. Scientific, Inc. (San Diego, CA) (H-1150) as WO03/027275. CGP60474, a phenylaminopyrimidine 21; WO95/09853, Zimmermannet al., Sep. 21, 1994 Thiazolopyrimidine 2 Attaby et al., Z.Naturforsch. 54b, 788-798 (1999) Diarylurea Honma, T. et al., J. Med.Chem., 44, 4628-4640 (2001), Honma, T. et al., J. Med. Chem., 44,4615-4627 (2001). (2R)-2,5-Dihydro-4-hydroxy-2-[(4-hydroxy-3-(3-Kitagawa, M. et al., Oncogene, 8, 2425-2432methyl-2-butenyl)phenyl)methyl]-3-(4- (1993).hydroxyphenyl)-5-oxo-2-furancarboxylic acid methyl ester having amolecular formula of C₂₄H₂₄O₇ available from Sigma-Aldrich under thetrade name Butyrolactone-I (B7930) Aloisine A, Cat. No. 128125(Calbiochem, San Mettey et al., J. Med. Chem. 2003, 46, 222-236 Diego,CA)

In a preferred embodiment, the c-MAF inhibitory, agents are used for thetreatment and/or prevention of bone metastasis. In a yet more preferredembodiment, the bone metastasis is osteolytic metastasis.

The c-MAF inhibitory agents are typically administered in combinationwith a pharmaceutically acceptable carrier.

The term “carrier” refers to a diluent or an excipient whereby theactive ingredient is administered. Such pharmaceutical carriers can besterile liquids such as water and oil, including those of a petroleum,animal, plant or synthetic origin such peanut oil, soy oil, mineral oil,sesame oil and the like. Water or aqueous saline solutions and aqueousdextrose and glycerol solutions, particularly for injectable solutions,are preferably used as carriers. Suitable pharmaceutical carriers aredescribed in “Remington's Pharmaceutical Sciences” by E. W. Martin,1995. Preferably, the carriers of the invention are approved by thestate or federal government regulatory agency or are listed in theUnited States Pharmacopeia or other pharmacopeia generally recognizedfor use thereof in animals and more particularly in human beings.

The carriers and auxiliary substances necessary for manufacturing thedesired pharmaceutical dosage form of the pharmaceutical composition ofthe invention will depend, among other factors, on the pharmaceuticaldosage form chosen. Said pharmaceutical dosage forms of thepharmaceutical composition will be manufactured according to theconventional methods known by the person skilled in the art. A review ofthe different methods for administering active ingredients, excipientsto be used and processes for producing them can be found in “Tratado deFarmacia Galenica”, C. Fauli i Trillo, Luzán 5, S. A. 1993 Edition.Examples of pharmaceutical compositions include any solid composition(tablets, pills, capsules, granules, etc.) or liquid composition(solutions, suspensions or emulsions) for oral, topical or parenteraladministration. Furthermore, the pharmaceutical composition may contain,as deemed necessary, stabilizers, suspensions, preservatives,surfactants and the like.

For use in medicine, the c-MAF inhibitory agents can be found in theform of a prodrug, salt, solvate or clathrate, either isolated or incombination with additional active agents and can be formulated togetherwith a pharmaceutically acceptable excipient. Excipients preferred foruse thereof in the present invention include sugars, starches,celluloses, rubbers and proteins. In a particular embodiment, thepharmaceutical composition of the invention will be formulated in asolid pharmaceutical, dosage form (for example tablets, capsules, pills,granules, suppositories, sterile crystal or amorphous solids that can bereconstituted to provide liquid forms etc.), liquid pharmaceuticaldosage form (for example solutions, suspensions, emulsions, elixirs,lotions, ointments, etc.) or semisolid pharmaceutical dosage form (gels,ointments, creams and the like). The pharmaceutical compositions of theinvention can be administered by any route, including but not limited tothe oral route, intravenous route, intramuscular route, intraarterialroute, intramedularry route, intrathecal route, intraventricular router,transdermal route, subcutaneous route, intraperitoneal route, intranasalroute, enteric route, topical route, sublingual route or rectal route. Areview of the different ways for administering active ingredients of theexcipients to be used and of the manufacturing processes thereof can befound in Tratado de Farmacia Galenica, C. Fauli i Trillo, Luzán 5, S.A., 1993 Edition and in Remington's Pharmaceutical Sciences (A. R.Gennaro, Ed.), 20^(th) edition, Williams & Wilkins PA, USA (2000).Examples of pharmaceutically acceptable carriers are known in the stateof art and include phosphate buffered saline solutions, water, emulsionssuch as oil/water, emulsions, different types of wetting agents, sterilesolutions, etc. The compositions comprising said carriers can beformulated by conventional processes known in the state of the art.

In the event that nucleic acids (siRNA, polynucleotides encoding siRNAor shRNA or polynucleotides encoding negative c-MAF dominants) areadministered the invention contemplates pharmaceutical compositionsparticularly prepared for administering said nucleic acids. Thepharmaceutical compositions can comprise said naked nucleic acids, i.e.,in the absence of compounds protecting the nucleic acids fromdegradation by the nucleases of the body, which entails the advantagethat the toxicity associated with the reagents used for transaction iseliminated. Administration routes suitable for naked compounds includethe intravascular route, intratumor route, intracranial route,intraperitoneal route, intrasplenic route, intramuscular route,subretinal route, subcutaneous route, mucosal route, topical route andoral route (Templeton, 2002, DNA Cell Biol., 21:857-867). Alternatively,the nucleic acids can be administered forming part of liposomesconjugated to cholesterol or conjugated to compounds capable ofpromoting the translocation through cell membranes such as the Tatpeptide derived from the HIV-1 TAT protein, the third helix of thehomeodomain of the D. melanogaster antennapedia protein, the herpessimplex virus VP22 protein, arginine oligomers and peptides as describedin WO07069090 (Lindgren, A. et. al., 2000, Trends Pharmacol. Sci,21:99-103 Schwarze, S. R. et al., 2000, Trends Pharmacol. Sci.,21:45-48, Lundberg, M et al., 2003, Mol Therapy 8:143-150 and Snyder, E.L. and Dowdy, S. F., 2004, Pharm. Res. 21:389-393). Alternatively, thepolynucleotide can be administered forming part of a plasmid vector orviral vector, preferably adenovirus-based vectors, in adeno-associatedviruses or in retroviruses such as viruses based on murine leukemiavirus (MLV) or on lentivirus (HIV, FIV, EIAV).

The c-MAF inhibitory agents or the pharmaceutical compositionscontaining them can be administered at a dose of less than 10 mg perkilogram of body weight preferably less than 5, 2, 1, 0.5, 0.1, 0.05,0.01, 0.005, 0.001, 0.0005, 0.0001, 0.00005 or 0.00001 mg per kg of bodyweight. The unit-dose can be administered by injection, inhalation ortopical administration.

The dose depends on the severity and the response of the condition to betreated and it may vary between several days and months or until thecondition subsides. The optimal dosage can be determined by periodicallymeasuring the concentrations of the agent in the body of the patient.The optimal dose can be determined from the EC50 values obtained bymeans of previous in vitro or in vivo assays in animal models. The unitdose can be administered once a day or less than once a day, preferablyless than once every 2, 4, 8or 30 days. Alternatively, it is possible toadminister a starting dose followed by one or several maintenance doses,generally of a lesser, amount than the starting dose. The maintenanceregimen may involve treating the patient with a dose ranging between0.01 μg and 1.4 mg/kg of body weight per day, for example 10, 1, 0.1,0.01, 0.001, or 0.00001 mg per kg of body weight per day. Themaintenance doses are preferably administered at the most once every 5,10 or 30 days. The treatment must be continued for a time that will varyaccording to the type of disorder the patient suffers, the severitythereof and the condition of the patient. After treatment, the progressof the patient must be monitored to determine if the dose should beincreased in the event that the disease does not respond to thetreatment or the dose is reduced if an improvement of the disease isobserved or if unwanted side effects are observed.

Treatment or Prevention of the Bone Degradation in Lung Cancer Patientswith Bone Metastasis, having Elevated c-MAF Levels

The present inventors have determined that the c-MAF levels are elevatedin metastasis, and in a preferred embodiment, bone metastasis. Patientssuffering lung cancer which has metastasized in bone and in which thereare elevated c-MAF levels in said metastasis may benefit particularlyfrom therapies aimed at preventing the bone degradation caused by theincreased osteoclastic activity.

Thus, in another aspect, the invention relates to the use of an agentfor avoiding or preventing bone degradation in the preparation of amedicinal product for the prevention and/or the treatment of the bonemetastasis in a subject buffering lung cancer and having elevated c-MAFlevels in a metastatic tumor tissue sample with respect to a controlsample.

Alternatively, the invention relates to an agent for avoiding orpreventing bone degradation for use in the prevention and/or thetreatment of the bone, metastasis in a subject suffering lung cancer andhas elevated c-MAF levels in a metastatic tumor tissue sample withrespect to a control sample.

Alternatively, the invention relates to a method of prevention and/ortreatment of the degradation in a subject suffering lung cancer and haselevated c-MAF levels in a metastatic tumor tissue sample with respectto a control sample, which comprises administering an agent for avoidingor preventing bone degradation to said subject.

In a particular embodiment, the bone metastasis is osteolyticmetastasis. In another particular embodiment, the lung cancer is NSCLC.

The terms and expressions, “subject”, “lung cancer”, “tumor sample”,“metastasis”, “c-MAF gene”, “increased or elevated expression levels”and “control sample” have been described in detail in relation with thefirst method of the invention and are equally applicable to the agentfor avoiding or preventing bone degradation.

Agents capable of avoiding or preventing bone degradation suitable forthe therapeutic method described in the present invention have beendescribed in detail above in the context of the customized therapymethod.

The reference or control sample is a tumor sample of a subject, withlung cancer who has not suffered metastasis or that correspond to themedian value of the c-MAF gene expression levels measured in a tumortissue collection in biopsy samples, of subjects with lung cancer whohave not suffered metastasis.

Methods for determining or quantifying if the c-MAF levels are elevatedwith respect to a control sample have been described in detail inrelation with the first method of the invention and are equallyapplicable to the agent for avoiding or preventing bone degradation.

Alternatively a combined treatment can be carried out, in which morethan one agent for avoiding or preventing bone degradation from thosementioned above are combined to treat and/or prevent the metastasis orsaid agents can be combined with other supplements, such as calcium orvitamin D or with a hormone.

The agents for avoiding or preventing bone degradation are typicallyadministered in combination with a pharmaceutically acceptable carrier.The term “carrier” and the types of carriers have been defined above forthe c-MAF inhibitory agents as well as the form and the dose in whichthey can be administered and are equally applicable to the agent foravoiding or preventing bone degradation.

Kits of the Invention

In another aspect, the invention relates to a kit for predicting bonemetastasis of a lung cancer, in a subject suffering from said cancer,the kit comprising: a) means for quantifying the expression level ofc-MAF in a sample of said subject; and b) means for comparing thequantified level of expression of c-MAF in said sample to a referencec-MAF expression level.

In another aspect, the invention relates to a kit for predicting theclinical outcome of a subject suffering from bone metastasis from a lungcancer, the kit comprising: a) means for quantifying the expressionlevel of c-MAF in a sample of said subject; and b) means for comparingthe quantified expression level of c-MAF in said sample to a referencec-MAF expression level.

In another aspect the invention relates to a kit for determining atherapy for a subject suffering from lung cancer, the kit comprising: a)means for quantifying the expression level of c-MAF in a sample of saidsubject; b) means for comparing the quantified expression level of c-MAFin said sample to a reference c-MAF expression level; and c) means fordetermining a therapy for preventing and/or reducing bone metastasis insaid subject based on the comparison of the quantified expression levelto the reference expression level; d) means for excluding a therapy forpreventing and/or reducing bone metastasis in said subject based on thecomparison of the quantified expression level to the referenceexpression level.

In another aspect the invention relates to a kit comprising: i) areagent for quantifying the expression level of c-MAF in a sample of asubject suffering from lung cancer, and ii) one or more c-MAF geneexpression level indices that have been predetermined to correlate withthe risk of bone metastasis.

Means for quantifying the expression level of c-MAF in a sample of saidsubject have been previously described in detail including 16q23 and16q22-24 locus amplification and translocation.

In a preferred embodiment, means for quantifying expression comprise aset of probes and/or primers that specifically bind and/or amplify thec-MAF gene.

In particular embodiment the lung cancer is SCLC or NSCLC cancer.

In particular embodiment the kit is applied, but not limited, to a lungcancer biopsy, circulating lung cancer cell, circulating lung tumor DNA.

All the particular embodiments of the methods of the present inventionare applicable to the kits of the invention and to their uses.

Method for Typing a Sample of a Subject Suffering Lung Cancer.

In another aspect, the invention relates to an in vitro method fortyping a sample of a subject suffering from lung cancer, the methodcomprising:

-   -   a) providing a sample from said subject;    -   b) quantifying the expression level of c-MAF in said sample;    -   c) typing said sample by comparing the quantified, expression        level of c-MAF to a predetermined reference level of c-MAF        expression;        wherein said typing provides prognostic information related to        the risk of bone metastasis in said subject.

Means for quantifying the expression level of c-MAF in a sample of saidsubject. have been previously described in detail including 16q23 and16q22-24 locus amplification and translocation. In particular embodimentthe lung cancer is SCLC or NSCLC. In a preferred embodiment the sampleis a tumor tissue sample, a circulating tumor cell or a circulatingtumor DNA.

Method for Classifying a Subject Suffering from Lung Cancer

In another aspect, the invention relates to a method for classifying asubject suffering from lung cancer into a cohort, comprising: a)determining the expression level of c-MAF in a sample of said subject;b) comparing the expression level of c-MAF in said sample to apredetermined reference level of c-MAF expression; and c) classifyingsaid subject into a cohort based on said expression level of c-MAF inthe sample.

Means for quantifying the expression level of c-MAF in a sample of saidsubject have been: previously described in detail including 16q23 and16q22-24 locus amplification and translocation.

In particular embodiment the lung cancer is a SCLC or a NSCLC. In apreferred embodiment the sample is a tumor tissue sample, a circulatingtumor cell or a circulating DNA. In a preferred embodiment said cohortcomprises at least one other individual who has been determined to havea comparable expression level of c-MAF in comparison to said referenceexpression level In another preferred embodiment said expression levelof c-MAF in said sample is increased relative to said predeterminedreference level, and wherein the members of the cohort are classified ashaving increased risk of bone metastasis. In another preferredembodiment said cohort is for conducting a clinical trial. In apreferred embodiment, the sample is a tumor tissue sample.

The following examples illustrate the invention and do not limit thescope thereof.

EXAMPLES Example 1

Clinical Relevance and Prognostic Value of c-MAF and 16q22-24 toIdentify Patents at Risk of Metastasis, in Particular bone Metastasis

c-MAF was tested in different lung cancer patient sample cohorts. Thefirst including gene expression profiles and clinical annotations formetastasis, that contain the transcriptome of lung cancer primary,tumors and a second cohort based on lung tumor biopsies in the form of atissue microarray and the clinical annotations for time to bonemetastasis and overall survival. These tumors are representative of alllung cancer subtypes and stages. In the first cohort, the expression ofc-MAF gene is correlated with clinical parameters including time tometastasis. Similarly, the second set (n=74) of lung primary tumorbiopsies for which the clinical annotation for bone relapse and overallsurvival post primary tumor diagnosis was used to determine, the levelsof c-MAF protein expression and 16q23 genomic gain byimmunohistochemistry and fluorescence in situ hybridization using ac-MAF specific antibody and a c-MAF locus specific FISH probe and theassociation between the levels of c-MAF expression and 16q23 genomicgain and risk of bone relapse is established.

Explanation in Detail: Patient Cohort I

We did the appropriate statistical analysis to see if MAF expression inthese tumors correlates with metastasis. We used a cohort of lungprimary tumors, including EGFR and KRAS mutants and SCLC and NSCLC. Thepatients' information was extracted from:

-   -   Chitale D, Gong Y, Taylor B S, Broderick S, Brennan C, Somwar R,        Golas B, Wang L, Motoi N, Szoke J. Reinersman J M, Major J,        Sander C, Seshan, V E, Zakowski M F, Rusch V, Pao W, Gerald W,        Ladanyi M. “An integrated genomic analysis of lung cancer        reveals loss of DUSP4 in EGFR-mutant tumors.” Oncogene. 2009;        28: 2773-83.        All statistical analyses were performed in R using Bioconductor        (Gentleman et al. (2004)).        We adjusted Cox Proportional Hazards Models (using the R        function coxph from Packaged survival) to see if we could        explain metastasis phenotype through MAF expression. C-MAF had a        statistically significant effect (Using three independent probes        specific for c-MAF in affymetrix arrays). Gene expression of        c-MAF in lung primary tumors correlated significantly with        metastasis as its observed in kaplan meier plot and is        highlighted by the HR and the p-value obtained (FIG. 1) Patient        cohort II

Similarly, a dataset of (74) lung primary tumors for which the clinicalannotation for bone relapse post primary tumor diagnosis is available issecured, the levels of c-MAF are determined by immunohistochemistryusing a c-MAF specific antibody and the association between the levelsof c-MAF expression and risk of bone relapse is established. The samplesfrom the second paraffin embedded primary tumor tissue from lung cancerpatients. These samples were collected by Vall d'Hebron OncologyInstitute during its clinical practice together with the relevantclinical data needed and the approval of the clinical committee.

The samples were selected fulfill the following requests:

9 samples belonged to patients with local disease (M0) at diagnosis witha confirmed bone relapse at any time of follow-up.

49 samples belonged to patients at diagnosis that remain metastasis freeafter at least 5 years.

The remaining 16 tumors are from patients M0 at diagnosis that latterhad a relapse in any location other than bone.

Example 2

c-MAF Gene Expression is Associated with Risk of Metastasis, inParticular Bone Metastasis, in Lung Cancer

Expression of c-MAF bone metastasis genes was correlated with clinicalparameters including metastasis using the above described lung cancerexpression data set. As described in FIG. 1, c-MAF expression wasassociated to high risk of metastasis. Following a standard statisticalanalysis we showed that MAF expression in these tumors significantlycorrelates with metastasis. A cohort of lung primary tumors, includingEGFR and KRAS mutants and SCLC and NSCLG were used. All statisticalanalyses were performed using Bioconductor (Gentleman et al. (2004)).

The Cox Proportional Hazards Models were adjusted (using the R functioncoxph from Packaged survival) to see if we could explain metastasisphenotype through MAF expression. c-MAF had a statistically significanteffect (Using three independent probes specific for c-MAF in affymetrixarrays). Gene expression of c-MAF in lung primary tumors correlatedsignificantly with metastasis as its observed in kaplan meier plot andis highlighted by the HR and the p-value obtained (FIG. 1). Tumors (wereclassified according to three groups of c-MAF expression, high (>+1standard deviation of the mean), medium (<+1 standard deviation of themean and >−1 standard deviation of the mean) and low (<−1 standarddeviation of the mean).

Example 3 c-MAF Expression is Associated with Risk of Bone Metastasis

Immunohistochemistry Analysis

cMAF immunostaining was performed on plus charged glass slides in a DakoLink platform. After deparaffination, antigen retrieval was performed.Endogenous peroxidase is quenched. A anti-c-MAF antibody was used,followed by incubation with a secondary antibody coupled withpreoxidase. C-MAF immunostaining is scored by a computerizedmeasurement.

Prognostic and Predictive Value of c-MAF for Metastasis and BoneMetastasis in Lung Cancer

The prognostic and predictive value of c-MAF expression for metastasisof lung cancer was evaluated. C-MAF protein levels were determined byimmunohistochemistry (IHC). MAF immunostaining was scored by acomputerized measurement. Nine representative images from each specimenwere acquired at 10-nm wavelength intervals between 420 and 700 nm,using a DM2000 Leica microscope equipped with the Nuance FXMultispectral Imaging System (CRI Inc). Before acquiring a spectraldataset of an image, an autoexposure routine was performed while imaginga blank area of slides to determine, the exposure time necessary toapproximately 90% fill the device wells at each wavelength to compensatefor variations in source intensity, filter transmission efficiency andcamera sensitivity. A library of pure DAB and Hematoxylin dye colors wascreated and used to unmix the colors using the Nuance 1.6.4 software. Acube (stack of images, taken at the different wavelengths) of referencewas then acquired for each new case, followed by spectral imaging ofthree representative tissue fields using the same exposure times. Afterdeconvolution of the images, the spectral data was flat fielded tocompensate for unevenness in illumination and background was filtered.The positive signals were converted from transmission to optical densityunits by taking the negative log of the ratio of the sample divided bythe reference cube using a Beer law conversion. A computer-aidedthreshold was set, which creates a pseudo-color image that highlightsall of the positive signals. Analysis yielded quantitative data of c-MAFfrom the average intensity of regions of interest. Only the nuclei ofepithelial cells (normal and malignant), but not stromal cells orlymphocytes, were automatically detected by setting distinct sizethreshold and confirmed by a pathologist. Each case was calculated forthe mean value of the signal intensity of all regions of interest forstatistical analysis. The output of the computerized measurementproduced a continuous data ranging from 1160 to 99760 optical densityunits (O.D.) for c-MAF expression. The cut off (20000 O.D.) for high anlow expression was determined based on a receiving operating curve asper standard procedures (AUC 0.802). The results are summarized in Table3.

TABLE 3 Bone metastasis NO Yes c-MAF <=20,000 OD 61 2 >20,000 OD 4 7

Based on this values the odds ratio of risk of suffering bone metastasisin the c-MAF high group versus the low was OR (bone metastasis at anytime)=53.37 (95% C.I. 8.24-345.92)

Next the time to bone progression of the overall population of the studywas confirmed (FIG. 2a ) and next the probability of bone metastasis atany time survival-free of the two groups was evaluated (FIG. 2b ).Interestingly, in the cohort of study, the c-MAF high expressing tumorshave a smaller overall survival than those expressing low levels.

Based on the second cohort analyzed, diagnostic clinical, features wereextracted. C-MAF high level expression predicts bone metastasis with asensitivity of 0.778, a specificity of 0.938, a positive predictivevalue of 63.6%, and a negative predictive value of 96.8%.

The c-MAF gene or protein expression in lung tumors correlatessignificantly with metastasis and bone metastasis at any time.

Example 4

16q22-q24 (Includes c-MAF Gene) Chromosome Amplification Predicts and isa Prognostic Indicator of Risk of Metastasis, in Particular BoneMetastasis, in Lung Cancer

Next, we determined whether a gain in chr16q22-q24, which includes c-MAFgene locus, is associated with risk of bone metastasis in lung cancerpatients. To this end a 16q23 14q32 dual fluorescence in situhybridization (FISH) probe that identifies chr16q22-q24 amplificationswas used to measure the number of copies of the 16q22-24 region. The14q32 probe was also used to normalize tumor polyploidy.

The prognostic and predictive value of 16q22-24 CNA gain associationwith bone metastasis in lung cancer was evaluated. 16q23 and 14q32copies were determined by FISH. The slides were incubated with 16q23 MAFand 14q32IGH probe mixture. This SpectrumOrange probe flanks the MAFgene region and is composed of two segments that are each approximately350 kb with an approximately 2.2 Mb gap. The centromeric segment islocated at chr16:75729985-76079705 (March 2006 assembly, UCSC GenomeBrowser) and the telomeric segment is located at chr16:78290003-78635873(March 2006 assembly, UCSC Genome Browser). This probe flanks five genesVAT1L, CLEC3A, WWOX, 5srRNA and MAF (ordered from centromer to telomer).In parallel, a CEP 16 (centromeric chr. 16) probe (Abbot) was used todetermine the CNA. CEP 16 (16q11.2) (Abbot) probe was used to score16q23 CNA. DAPI counterstain was applied and images were acquired with afluorescence microscope.

The results are summarized in Tables 4 and 5.

TABLE 4 A tumor will be positive for a 16q22-24 gain based on a cutoff >= to 2.5 copies of the 16q23 Bone metastasis NO Yes 16q23 <=2.5 139 FISH >2.5 44 0

TABLE 5 A tumor will be positive for a 16q22-24 CAN gain based on a cutoff >= to 1.5 copies of the 16q23 normalized by number of the copies ofthe 14q23. Bone metastasis NO Yes 16q23 <=1.5 4 9 CNA >1.5 53 0

Based on these values the odds ratio of risk of suffering bonemetastasis in the 16q22-24 gain or CNA gain positive group versus thenegative was calculated. Since there were no patients with bonemetastasis that score negative for the determination and in order toavoid errors on the estimation, as per standard statistical procedures(Glas, A. S. et al., 2003, Journal of Clinical Epidemiology 56,1129-1135), 0.5 units were added to each value and recalculated theparameter. Based on this estimation, the OR for the 16q22-24 gainpositive patients to suffer a bone metastasis was 62.63 (95% CI3.42-1147.78), and the OR for the 16q22-24 CNA gain normalized using14q32 positive patients versus the control was 225.89 (95% CI11.22-4546.99). The small size of the cohort made the estimatesimprecise but within a clinically relevant OR of least 3.42 and 11.22 ineach case.

Based on the data using FISH in order to measure 16q23 gains, diagnosticclinical features were extracted. 16q22-24 gain (>=2.5 copies per cell)predicts lung cancer patients at high risk of bone metastasis with asensitivity of 1.00, a specificity of 0.772, a positive predictive valueof 40.9%, and a negative predictive value of 100.0%.

TABLE 6 Diagnostic clinical features. C.I. 95% Sensitivity 100.0%70.1%-100.0% Specificity 77.2% 64.8%-86.2%  PPV 40.9% 23.3%-61.3%  NPV100.0% 92.0%-100.0%

Based on the data using FISH in order to measure 16q23 gains normalizedto 14q32 copies, diagnostic clinical features were extracted. 16q22-24CNA gain (>=1.5 16q23 copies per cell normalized to 14q32) predicts lungcancer risk of bone metastasis with a sensitivity of 1.00, a specificityof 0.930, a positive predictive value of 69.2%, and : a negativepredictive value of 100.0%.

TABLE 7 Diagnostic clinical features CI 95% Sensitivity 100.0%70.1%-100.0% Specificity 93.0% 83.3%-97.2%  PPV 69.2% 42.4%-87.3%  NPV100.0% 93.2%-100.0%

The 16q22-24, and particularly 16q23, amplification/gain or CNA gain inlung tumors strongly predicts and is associated with risk of bonemetastasis at any time.

1-37. (canceled)
 38. An in vitro method for the diagnosis of metastasisand/or recurrence in a subject with lung cancer and/or for the prognosisof the tendency to develop metastasis and/or recurrence in a subjectwith lung cancer, and for the treatment of said subject to inhibit saidmetastasis and/or recurrence and/or to avoid or inhibit bonedegradation, comprising, (i) quantifying the c-MAF gene expressionlevel, amplification or copy number in a tumor sample of said subject;and, (iii) determining that the subject has an increase in theexpression level, copy number or amplification of the c-MAF gene in thetumor sample with respect to the expression level, copy number oramplification of the c-MAF gene in the control sample, and (iv)administering a therapeutically effective amount of a c-MAF inhibitor, atherapy aiming to inhibit and/or treat bone metastasis selected from thegroup consisting of an mTor inhibitor, a Src kinase inhibitor, a COX-2inhibitor, a CCR5 antagonist and/or Radium-223, and/or an agent capableof avoiding, inhibiting and/or treating bone degradation to saidsubject.
 39. The method according to claim 38, wherein the metastasis isbone metastasis.
 40. The method according to claim 39, wherein the bonemetastasis is osteolytic bone metastasis.
 41. The method according toclaim 39, wherein the expression level, copy number or amplification isquantified by means of quantitative polymerase chain reaction (PCR) or aDNA or RNA array, nucleotide hybridization technique, western blot, insitu hybridization, ELISA, immunohistochemistry or a protein array. 42.The method according to claim 39, wherein the agent capable of avoiding,inhibiting and/or treating bone degradation is selected from the groupconsisting of: a bisphosphonate, a RANKL inhibitor, a PTH or a PTHLHinhibitor, a PRG analog, strontium ranelate, a DKK-1 inhibitor, a dualMET and VEGFR2 inhibitor, an estrogen receptor modulator, calcitonin,Radium-223, a cathepsin K inhibitor and combinations thereof.
 43. Themethod according to claim 42, wherein the RANKL inhibitor is selectedfrom the group consisting of: a RANKL specific antibody, a RANKLspecific nanobody, and osteoprotegerin.
 44. The method according toclaim 42, wherein the RANKL inhibitor is the RANKL specific antibodydenosumab, the bisphosphonate is zoledronic acid, the RANKL inhibitor isthe RANKL specific nanobody ALX-0141 or the dual MET and VEGFR2inhibitor is Cabozantinib.
 45. An in vitro method for diagnosingmetastasis and/or recurrence in a subject with lung cancer and/or forthe prognosis of the tendency to develop metastasis and/or recurrenceand for treating said subject to inhibit said metastasis or recurrence,comprising: (i) detecting c-MAF amplification in a tumor sample of saidsubject relative to a reference gene copy number, wherein theamplification of the c-MAF gene is determined by means of determiningthe amplification of the locus 16q22-q24, wherein an amplification ofthe c-MAF gene with respect to said reference gene copy number isindicative of the presence of metastasis or recurrence or an increasedrisk of developing metastasis or recurrence, and (ii) administering atherapeutically effective amount of a c-MAF inhibitor, a therapy aimingto inhibit and/or treat bone metastasis selected from the groupconsisting of an mTor inhibitor, a Src kinase inhibitor, a COX-2inhibitor, a CCR5 antagonist and/or Radium-223, and/or an agent capableof avoiding, inhibiting and/or treating bone degradation to said subjectwith an increase in the amplification of the c-MAF gene with respect toreference gene copy number.
 46. The method according to claim 45,wherein the metastasis is bone metastasis.
 47. The method according toclaim 46, wherein the bone metastasis is osteolytic bone metastasis. 48.The method according to claim 45, wherein the amplification isquantified by means of in situ hybridization or PCR.
 49. The methodaccording to claim 48, wherein the amplification of the c-MAF gene isdetermined by means of determining the amplification of the locus16q22-q24.
 50. The method according to claim 45, wherein the agentcapable of avoiding, inhibiting and/or treating bone degradation isselected from the group consisting of: a bisphosphonate, a RANKLinhibitor, a PTH or a PTHLH inhibitor, a PRG analog, strontium ranelate,a DKK-1 inhibitor, a dual MET and VEGFR2 inhibitor, an estrogen receptormodulator, calcitonin, Radium-223, a cathepsin K inhibitor andcombinations thereof.
 51. The method according to claim 50, wherein theRANKL inhibitor is selected from the group consisting of: a RANKLspecific antibody, a RANKL specific nanobody, and osteoprotegerin. 52.The method according to claim 50, wherein the RANKL inhibitor is theRANKL specific antibody denosumab, the bisphosphonate is zoledronicacid, the RANKL inhibitor is the RANKL specific nanobody ALX-0141 or thedual MET and VEGFR2 inhibitor is Cabozantinib.
 53. An in vitro methodfor typing a sample of a subject suffering from lung cancer, the methodcomprising: a) providing a sample from said subject; b) quantifying theexpression level, amplification or copy number of c-MAF in said sample;c) typing said sample by comparing the quantified expression level,amplification, or copy number of c-MAF to a predetermined referenceexpression level, degree of amplification or copy number; wherein saidtyping provides prognostic information related to the risk of bonemetastasis in said subject.
 54. A method of classifying a subjectsuffering from lung cancer into a cohort for a clinical trial,comprising: a) determining the expression level, copy number oramplification of c-MAF in a sample of said subject; b) comparing theexpression level, copy number or amplification of c-MAF in said sampleto a predetermined reference; and c) classifying said subject into acohort based on said expression level, copy number or amplification ofc-MAF in the sample.